Cas 9 retroviral integrase systems for targeted incorporation of a dna sequence into a genome of a cell

CROSS REFERENCE TO RELATED APPLICATION

0001This application claims the benefit of <patcit id="pcit0001" dnum="US62140454A"><text>United States Provisional Application No. 62,140,454, filed March 31, 2015</text></patcit>, <patcit id="pcit0002" dnum="US62210451A"><text>United States Provisional Application No. 62,210,451, filed August 27, 2015</text></patcit>, and <patcit id="pcit0003" dnum="US62240359A"><text>United States Provisional Application No. 62,240,359 filed October 12, 2015</text></patcit>, the entire contents of each are incorporated by reference for all purposes.

INTRODUCTION

0002The instant disclosure relates to the use of engineered proteins with DNA binding proteins exhibiting genome specificity such as Cas9 (CRISPR (clustered regularly interspaced short palindromic repeats) protein), TALE and Zinc finger proteins attached by a linker with a viral integrases (e.g. HIV or MMTV integrases) or a recombinase in order to deliver a DNA sequence of interest (or gene of interest) to a targeted site in a genome of a cell or organism. The use of a Cas9 that is inactive for its function in cutting DNA will allow us to use the Cas9 proteins ability to target DNA by the use of RNA guides (gRNA) without causing DNA breaks as intended in other systems for homologous recombination. The use of zinc finger proteins or TALE (engineered proteins that bind specific sequences of DNA) attached to the viral integrase or the recombinase is also disclosed. The system may be used for laboratory and therapeutic purposes. For example, donor DNA containing the gene(s) of interested can be easily introduced into host genome without the potential of off target cuts through conventional methods. Donor DNA can be engineered to facilitate "knock out" strategies as well. A new strategy for improving the specificity of Cas9 targeting is also discussed. This strategy uses surface bound dCas9 (Cas9 that is inactive for its DNA cutting ability) along with guide RNAs and genomic DNA in an assay to find which guide RNAs provide specific targeting of the Cas9. This will be especially important in in vivo applications of CRISPR/Cas9 and overcome limitations of the current in silico prediction models, although it may also be used in conjunction with in silico prediction models to make an educated determination of which gRNAs will be used in the assay.

BACKGROUND

0003Current advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic/genomic factors that are associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), CRISPR/Cas9 or meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that will allow the incorporation of DNA sequences (including full gene sequences) into a specific location in a given genome. This will allow for the production of cell lines or transgenic organisms that express an engineered gene or for the replacement of dysfunctional genes in a subject in need thereof.

0004Integrases are viral proteins that allow for the insertion of viral nucleic acids into a host genome (mammalian, human, mouse, rat, monkey, frog, fish, plant (including crop plants and experimental plants like Arabidopsis), laboratory or biomedical cell lines or primary cell cultures, C. elegans, fly (Drosophila), etc.). Integrases use DNA binding proteins of the host to bring the integrase in association with the host genome in order to incorporate the viral nucleic acid sequence into the host genome. Integrases are found in a retrovirus such as HIV (human immunodeficiency virus). Integrases depend on sequences on viral genes to insert their genome into host DNA. <nplcit id="ncit0001" npl-type="s"><text>Leavitt et al (Journal of Biological Chemistry, 1993, volume 268, pages 2113-2119</text></nplcit>) examined the function of HIV1 integrase by using site directed mutagenesis and in vitro studies. Leavitt also indicates sequence of U5 and U3 HIV1 att sites that are important for the integration of HIV1 DNA (created after reverse transcription) into the host genome by the viral integrase.

0005The instant disclosure improves current genome editing technology by allowing one to specifically insert desired nucleic acid (DNA) sequences into the genome at specified locations in the genome. The recombinant engineered integrase (or recombinase) with DNA binding ability will bind a given DNA sequence in the genome and recognize a provided DNA sequence having integrase recognition domains (such as the HIV1 (or other retrovirus) att sites) and/or homology arms to insert the given nucleic acid sequence into the genome in a site specific manner. One aspect of the disclosure involves inserting DNA sequences of stop codons (UAA, UAG and/or UGA) just after the transcriptional start site of a gene. This will allow for effective inhibition of gene transcription in the genome of a cell or organism.

SUMMARY

0006The current disclosure links DNA targeting technologies including zinc finger proteins, TALEN and CRISPR/Cas9, or other CRISPR proteins like Cpf1 and the like, with retroviral integrases to form DNA targeting integrases. A gene of interest (GOI) may then be provided with the DNA targeting integrase so that it may be incorporated into the genome in a targeted manner. The GOI will be designed with homology arms to provide another level of specificity to its insertion in the genome.

0007The disclosure particularly relates to the use of a variant Cas9 that is inactive for cutting DNA for linking with a retroviral integrase.

0008The instant disclosure comprises a system comprising: A) a viral integrase (or a bacterial recombinase) covalently linked to a Cas protein (e.g. Cas9) that is, for example, inactive for DNA cutting ability. Alternatively, the viral integrase (or the recombinase) is covalently linked to a TALE protein or zinc finger proteins where these proteins are designed to target a specific sequence of DNA in a genome. This may be provided in an expression vector or as a purified protein; B) a gene of interest (or DNA sequence of interest) with or without homology arms to be incorporated into the desired genome. The GOI or DNA sequence of interest may be modified to be recognized by the viral integrase as needed. Other reagents needed for polynucleotide transfection and/or introduction of protein into cells. Assaying for off-target integration of DNA sequences. In one aspect, using a marker sequence engineered into the inserted DNA sequence.

0009Provided herein are nucleic acid constructs comprising in operable linkage: a) a first polynucleotide sequence encoding a Cas9, an inactive Cas9, or a Cpfl, or a portion thereof: b) a second polynucleotide sequence encoding an integrase, a recombinase, or a transposase, or a portion thereof; and c) a third polynucleotide sequence encoding a nucleic acid linker; wherein the first polynucleotide sequence comprises a 5' and a 3' end and the second polynucleotide sequence comprises a 5' and a 3' end, and the 3' end of the first polynucleotide is connected to the 5' end of the second polynucleotide by the nucleic acid linker, and the first and second polynucleotide are able to be expressed as a fusion protein in a cell or an organism. In some embodiments, the first polynucleotide sequence comprises any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 27-46, 49, 56, or 68, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the Cas9, an inactive Cas9, or a Cpf1comprises any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 50, 52, 69, 72-78, or 86-92, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the second polynucleotide sequence comprises any one of SEQ ID NOS: 15, 17, 19, 21, 23, 47, 55, 62, 64, 66, 70, or 79, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the integrase, recombinase, or transposase comprises any one of SEQ ID NOS: 16, 18, 20, 22, 24, 25, 26, 48, 63, 65, 67, 71, or 80, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Also described herein are organisms comprising the nucleic acid construct. Also described herein is an organism comprising the fusion protein wherein the organism has a modified genome.

0010Provided herein are organisms comprising: a) a first polynucleotide sequence encoding a Cas9, an inactive Cas9, or a Cpfl, or a portion thereof: b) a second polynucleotide sequence encoding an integrase, a recombinase, or a transposase, or a portion thereof; and c) a third polynucleotide sequence encoding a nucleic acid linker; wherein the first polynucleotide sequence comprises a 5' and a 3' end and the second polynucleotide sequence comprises a 5' and a 3' end, and the 3' end of the first polynucleotide is connected to the 5' end of the second polynucleotide by the nucleic acid linker, and the first and second polynucleotide are able to be expressed as a fusion protein in a cell or an organism.

0011Also provided herein are fusion proteins, comprising: a) a first protein that is a catalytically inactive Cas9, Cas9, a TALE protein, a Zinc finger protein, or a Cpf1 protein, wherein the first protein is targeted to a target DNA sequence; b) a second protein that is an integrase, a recombinase, or a transposase; and c) a linker linking the first protein to the second protein. In some embodiments, the second protein is an integrase; the integrase is an HIV1 integrase or a lentiviral integrase; the linker sequence is one or more amino acids in length; or the first protein is a catalytically inactive Cas9. In some embodiments, the linker sequence is 4-8 amino acids in length; the first protein is a TALE protein; or the first protein is a Zinc finger protein. In some embodiments, wherein the fusion protein comprises a TALE or a Zinc finger protein, the target DNA sequence is about 16 to about 24 base pairs in length. In some embodiments, the first protein is Cas9 or a catalytically inactive Cas9, and wherein one or more guide RNAs are used for targeting of a target DNA sequence of from about 16 to about 24 base pairs.

0012Also provided herein are methods of inserting a DNA sequence into genomic DNA, comprising: a) identifying a target sequence in the genomic DNA; b) designing a fusion protein according to claim 1 to bind to the target sequence in the genomic DNA; 3) designing a DNA sequence of interest to incorporate into the genomic DNA; and d) providing the fusion protein and the DNA sequence of interest to a cell or organism by techniques that allow for entry of the fusion protein and DNA sequence of interest into the cell or organism; wherein the DNA sequence of interest becomes integrated at the target sequence in the genomic DNA.

0013Also provided herein are nucleotide vectors, comprising: a) a first coding sequence for a first protein that is a Cas9, a catalytically inactive Cas9, a TALE protein, a Zinc finger protein, or a Cpf1 protein engineered to bind a target DNA sequence; b) a second coding sequence for a second protein that is an integrase, a recombinase, or a transposase; c) a DNA sequence between the first and second coding sequences that forms an amino acid linker between the first and second proteins; d) optionally an expressed DNA sequence of interest surrounded by att sites recognized by an integrase, and optionally one or more guide RNAs, wherein the first protein is targeted to a determined DNA sequence, and wherein the first protein is linked to the second protein by the amino acid linker sequence.

0014Provided herein are methods of inhibiting gene transcription in a cell or organism, comprising: a) identifying an ATG start codon in a gene; b) designing a fusion protein system with a fusion protein according to claim 1 to bind to a target sequence immediately after the ATG start codon of the gene; c) designing a DNA sequence of interest that is one or more consecutive stop codons; and d) providing the fusion protein and the DNA sequence of interest to a cell or organism by techniques that allow for entry of the fusion protein and DNA sequence of interest into the cell or organism; wherein the DNA sequence of interest becomes integrated at the target sequence in the genomic DNA; and wherein transcription of the gene is inhibited. In some embodiments, the second protein is a recombinase; the recombinase is a Cre recombinase or a modified version thereof, wherein the modified Cre recombinase has constitutive recombinase activity. In one embodiment, the vector further comprising a reverse transcriptase gene to be expressed in a cell.

0015Also provided herein are compositions, comprising a purified protein of a DNA binding protein/integrase fusion and an RNA from about 15 to about 100 base pairs in length, wherein the DNA binding protein is selected from Cas9, Cpfl, a TALEN and a Zinc finger protein engineered to a targeted DNA sequence in a genome, and wherein the integrase is a HIV integrase, lentiviral integrase, adenoviral integrase, a retroviral integrase, or a MMTV integrase.

BRIEF DESCRIPTION OF THE DRAWINGS

0016These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, appended claims and accompanying figures where: <ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001"><b>FIG. 1</b></figref> shows a) an exemplary catalytically inactive Cas9/HIV1 integrase fusion protein, b) an exemplary TALE/HIV1 integrase fusion protein, c) an exemplary zinc finger protein/HIV1 integrase fusion protein, and d) an exemplary Cas9/HIV1 integrase fusion protein designed to opposite sides of the DNA at the targeted site. Each of the fusion proteins binds to a specific target sequence of DNA. "ZnFn" is a Zinc finger protein. "Integrase" represents one integrase unit or two integrase units linked, for example, by a short amino acid linker. In some embodiments, the integrase may be replaced by a recombinase. Cas9 may be catalytically active or inactive.</li><li><figref idref="f0002"><b>FIG. 2</b></figref> shows a DNA plasmid system comprising, a vector comprising a catalytically inactive Cas9/integrase fusion protein, a vector comprising a DNA sequence of interest, and a vector comprising a reverse transcriptase. A guide RNA (gRNA) or RNAs may be provided separately. Another vector can be used to express a gRNA. "1 or 2" refers to one integrase or two integrases linked by, for example, an amino acid linker.</li><li><figref idref="f0003"><b>FIG. 3</b></figref> shows an exemplary DNA plasmid comprising a nucleiotide sequence catalytically inactive Cas9/integrase fusion protein, guide RNAs, a DNA (gene) sequence of interest, and a reverse transcriptase. Viral att sites can be provided to the DNA sequence of interest, allowing for incorporation of the integrase into the cell's genomic DNA. A guide RNA (gRNA) or RNAs may be provided separately. Another vector can be used to express a gRNA. "1 or 2" refers to one integrase or two integrases linked by, for example, an amino acid linker.</li><li><figref idref="f0004"><b>FIG. 4</b></figref> shows a flow diagram. One exemplary method of employing the vectors shown in <figref idref="f0002"><b>FIG. 2</b></figref> and <figref idref="f0003"><b>FIG. 3</b></figref> is shown in <figref idref="f0004"><b>FIG. 4</b></figref><b>,</b> and is as follows: 1) reverse transcriptase reverse transcribes the DNA sequence of interest with att sites expressed from the vector (alternatively a linear DNA with att sites is used), 2) fusion Cas9/integrase targets site on genomic DNA based on guide RNAs, 3) integrase recognizes att (LTR) sites on DNA sequence of interest and integrates the DNA into the genome at the targeted site, and 4) an assay (e.g. PCR (polymerase chain reaction) is conducted to check for proper insertion of DNA sequence of interest. An assay can be conducted to check for non-specific integration.</li><li><figref idref="f0005"><b>FIG. 5</b></figref> shows Abbie1 Gene Editing Targeting Exon 2 of Nrf2 Using Guide NrF2-sgRNA2 and sgRNA3.</li><li><figref idref="f0006"><b>FIG. 6</b></figref> shows theoretical data generated by Abbie1 gene editing.</li><li><figref idref="f0007"><b>FIG. 7</b></figref> shows A Abbie1 Gene Editing Targeting Exon 2 of Nrf2 Using Guide Nrf2-sgRNA 3.</li><li><figref idref="f0008"><b>FIG. 8</b></figref> shows Abbie1 Knock out of Nrf2 in pooled Hek293T Cells.</li><li><figref idref="f0009"><b>FIG. 9</b></figref> shows Abbie1 Knock out of Nrf2 in pooled Hek293T Cells.</li><li><figref idref="f0010"><b>FIG. 10</b></figref> shows Abbie1 Gene Editing Targeting CXCR4 Exon 2.</li><li><figref idref="f0011"><b>FIG. 11</b></figref> shows detection of ABBIE1 protein after isolation and purification [0028] from E coli. Coomassie stained gel.</li></ul>

DETAILED DESCRIPTION

0017The following detailed description is provided to aid those skilled in the art in practicing the present disclosure. Even so, this detailed description should not be construed to unduly limit the present disclosure as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present discovery.

0018As used in this disclosure and the appended claims, the singular forms "a", "an" and "the" include a plural reference unless the context clearly dictates otherwise. As used in this disclosure and the appended claims, the term "or" can be singular or inclusive. For example, A or B, can be A and B.

ENDOGENOUS

0019An endogenous nucleic acid, nucleotide, polypeptide, or protein as described herein is defined in relationship to the host organism. An endogenous nucleic acid, nucleotide, polypeptide, or protein is one that naturally occurs in the host organism.

EXOGENOUS

0020An exogenous nucleic acid, nucleotide, polypeptide, or protein as described herein is defined in relationship to the host organism. An exogenous nucleic acid, nucleotide, polypeptide, or protein is one that does not naturally occur in the host organism or is a different location in the host organism.

KNOCKOUT

0021A gene is considered knocked out when an exogenous nucleic acid is transformed into a host organism (e.g. by random insertion or homologous recombination) resulting in the disruption (e.g. by deletion, insertion) of the gene.

0022Upon knocking out a gene, the activity of the corresponding protein can be decreased. For example, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or 100%, as compared to the activity of the same protein wherein the gene has not been knocked out.

0023Upon knockout out of a gene, the transcription of the gene can be decreased, as compared to a gene that has not been knocked out, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or 100%.

MODIFIED

0024A modified organism is an organism that is different than an unmodified organism. For example, a modified organism can comprise a fusion protein of the disclosure that results in a knockout of a targeted gene sequence. A modified organism can have a modified genome.

0025A modified nucleic acid sequence or amino acid sequence is different than the unmodified nucleic acid sequence or amino acid sequence. For example, a nucleic acid sequence can have one or more nucleic acids inserted, deleted, or added. For example, an amino acid sequence can have one or more amino acids inserted, deleted, or added.

OPERABLY LINKED

0026In some embodiments, a vector comprises a polynucleotide operably linked to one or more control elements, such as a promoter and/or a transcription terminator. A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked sequences can be contiguous and, in the case of a secretory leader, contiguous and in reading phase.

HOST CELL OR HOST ORGANISM

0027A host cell can contain a polynucleotide encoding a polypeptide of the present disclosure. In some embodiments, a host cell is part of a multicellular organism. In other embodiments, a host cell is cultured as a unicellular organism.

0028Host organisms can include any suitable host, for example, a microorganism. Microorganisms which are useful for the methods described herein include, for example, bacteria (e.g., <i>E. coli</i>)<i>,</i> yeast (e.g., <i>Saccharomyces cerevisiae</i>), and plants. The organism can be prokaryotic or eukaryotic. The organism can be unicellular or multicellular.

0029The host cell can be prokaryotic. Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of <i>Escherichia coli, Lactobacillus</i> sp., <i>Salmonella</i> sp., and <i>Shigella</i> sp. (for example, as described in <nplcit id="ncit0002" npl-type="s"><text>Carrier et al. (1992) J. Immunol. 148:1176-1181</text></nplcit>; <patcit id="pcit0004" dnum="US6447784B"><text>U.S. Pat. No. 6,447,784</text></patcit>; and <nplcit id="ncit0003" npl-type="s"><text>Sizemore et al. (1995) Science 270:299-302</text></nplcit>). Examples of <i>Salmonella</i> strains which can be employed in the present disclosure include, but are not limited to, <i>Salmonella typhi</i> and S. <i>typhimurium</i>. Suitable <i>Shigella</i> strains include, but are not limited to, <i>Shigella flexneri, Shigella sonnei,</i> and <i>Shigella disenteriae.</i> Typically, the laboratory strain is one that is non-pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, <i>Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum,</i> and <i>Rhodococcus sp.</i>

0030In some embodiments, the host organism is eukaryotic. Suitable eukaryotic host cells include, but are not limited to, yeast cells, insect cells, plant cells, fungal cells, and algal cells.

POLYNUCLEOTIDES AND POLYPEPTIDES [NUCLEIC ACIDS AND PROTEINS]

0031The proteins of the present disclosure can be made by any method known in the art. The protein may be synthesized using either solid-phase peptide synthesis or by classical solution peptide synthesis also known as liquid-phase peptide synthesis. Using Val-Pro-Pro, Enalapril and Lisinopril as starting templates, several series of peptide analogs such as X-Pro-Pro, X-Ala-Pro, and X-Lys-Pro, wherein X represents any amino acid residue, may be synthesized using solid-phase or liquid-phase peptide synthesis. Methods for carrying out liquid phase synthesis of libraries of peptides and oligonucleotides coupled to a soluble oligomeric support have also been described. <nplcit id="ncit0004" npl-type="s"><text>Bayer, Ernst and Mutter, Manfred, Nature 237:512-513 (1972</text></nplcit>); <nplcit id="ncit0005" npl-type="s"><text>Bayer, Ernst, et al., J. Am. Chem. Soc. 96:7333-7336 (1974</text></nplcit>); <nplcit id="ncit0006" npl-type="s"><text>Bonora, Gian Maria, et al., Nucleic Acids Res. 18:3155-3159 (1990</text></nplcit>). Liquid phase synthetic methods have the advantage over solid phase synthetic methods in that liquid phase synthesis methods do not require a structure present on a first reactant which is suitable for attaching the reactant to the solid phase. Also, liquid phase synthesis methods do not require avoiding chemical conditions which may cleave the bond between the solid phase and the first reactant (or intermediate product). In addition, reactions in a homogeneous solution may give better yields and more complete reactions than those obtained in heterogeneous solid phase/liquid phase systems such as those present in solid phase synthesis.

0032In oligomer-supported liquid phase synthesis the growing product is attached to a large soluble polymeric group. The product from each step of the synthesis can then be separated from unreacted reactants based on the large difference in size between the relatively large polymer-attached product and the unreacted reactants. This permits reactions to take place in homogeneous solutions, and eliminates tedious purification steps associated with traditional liquid phase synthesis. Oligomer-supported liquid phase synthesis has also been adapted to automatic liquid phase synthesis of peptides. <nplcit id="ncit0007" npl-type="s"><text>Bayer, Ernst, et al., Peptides: Chemistry, Structure, Biology, 426-432</text></nplcit>.

0033For solid-phase peptide synthesis, the procedure entails the sequential assembly of the appropriate amino acids into a peptide of a desired sequence while the end of the growing peptide is linked to an insoluble support. Usually, the carboxyl terminus of the peptide is linked to a polymer from which it can be liberated upon treatment with a cleavage reagent. In a common method, an amino acid is bound to a resin particle, and the peptide generated in a stepwise manner by successive additions of protected amino acids to produce a chain of amino acids. Modifications of the technique described by Merrifield are commonly used. See, e.g., <nplcit id="ncit0008" npl-type="s"><text>Merrifield, J. Am. Chem. Soc. 96: 2989-93 (1964</text></nplcit>). In an automated solid-phase method, peptides are synthesized by loading the carboxy-terminal amino acid onto an organic linker (e.g., PAM, 4-oxymethylphenylacetamidomethyl), which is covalently attached to an insoluble polystyrene resin cross-linked with divinyl benzene. The terminal amine may be protected by blocking with t-butyloxycarbonyl. Hydroxyl- and carboxyl-groups are commonly protected by blocking with O-benzyl groups. Synthesis is accomplished in an automated peptide synthesizer, such as that available from Applied Biosystems (Foster City, Calif.). Following synthesis, the product may be removed from the resin. The blocking groups are removed by using hydrofluoric acid or trifluoromethyl sulfonic acid according to established methods. A routine synthesis may produce 0.5 mmole of peptide resin. Following cleavage and purification, a yield of approximately 60 to 70% is typically produced. Purification of the product peptides is accomplished by, for example, crystallizing the peptide from an organic solvent such as methyl-butyl ether, then dissolving in distilled water, and using dialysis (if the molecular weight of the subject peptide is greater than about 500 daltons) or reverse high pressure liquid chromatography (e.g., using a C¹⁸ column with 0.1% trifluoroacetic acid and acetonitrile as solvents) if the molecular weight of the peptide is less than 500 daltons. Purified peptide may be lyophilized and stored in a dry state until use. Analysis of the resulting peptides may be accomplished using the common methods of analytical high pressure liquid chromatography (HPLC) and electrospray mass spectrometry (ES-MS).

0034In other cases, a protein, for example, a protein is produced by recombinant methods. For production of any of the proteins described herein, host cells transformed with an expression vector containing the polynucleotide encoding such a protein can be used. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell such as a yeast, or the host can be a prokaryotic cell such as a bacterial cell. Introduction of the expression vector into the host cell can be accomplished by a variety of methods including calcium phosphate transfection, DEAE-dextran mediated transfection, polybrene, protoplast fusion, liposomes, direct microinjection into the nuclei, scrape loading, biolistic transformation and electroporation. Large scale production of proteins from recombinant organisms is a well established process practiced on a commercial scale and well within the capabilities of one skilled in the art.

CODON OPTIMIZATION

0035One or more codons of an encoding polynucleotide can be "biased" or "optimized" to reflect the codon usage of the host organism. For example, one or more codons of an encoding polynucleotide can be "biased" or "optimized" to reflect chloroplast codon usage or nuclear codon usage. Most amino acids are encoded by two or more different (degenerate) codons, and it is well recognized that various organisms utilize certain codons in preference to others. "Biased" or codon "optimized" can be used interchangeably throughout the specification. Codon bias can be variously skewed in different plants, including, for example, in alga as compared to tobacco. Generally, the codon bias selected reflects codon usage of the plant (or organelle therein) which is being transformed with the nucleic acids of the present disclosure.

0036A polynucleotide that is biased for a particular codon usage can be synthesized de novo, or can be genetically modified using routine recombinant DNA techniques, for example, by a site directed mutagenesis method, to change one or more codons such that they are biased for chloroplast codon usage.

PERCENT SEQUENCE IDENTITY

0037One example of an algorithm that is suitable for determining percent sequence identity or sequence similarity between nucleic acid or polypeptide sequences is the BLAST algorithm, which is described, e.g., in <nplcit id="ncit0009" npl-type="s"><text>Altschul et al., J. Mol. Biol. 215:403-410 (1990</text></nplcit>). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (as described, for example, in <nplcit id="ncit0010" npl-type="s"><text>Henikoff &amp; Henikoff (1989) Proc. Natl. Acad. Sci. USA, 89:10915</text></nplcit>). In addition to calculating percent sequence identity, the BLAST algorithm also can perform a statistical analysis of the similarity between two sequences (for example, as described in <nplcit id="ncit0011" npl-type="s"><text>Karlin &amp; Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993</text></nplcit>)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, less than about 0.01, or less than about 0.001.

0038The instant disclosure comprises a system comprising: A) A viral integrase (or a recombinase) covalently linked to a Cas protein (e.g. Cas9) that is, for example, inactive for DNA cutting ability. Alternatively, the viral integrase (or a bacterial or phage recombinase) is covalently linked to a TALE protein or zinc finger proteins where these proteins are designed to target a specific sequence of DNA in a genome.

0039This may be provided in an expression vector or as a purified protein. B) A gene of interest (or DNA sequence of interest) with or without homology arms to be incorporated into the desired genome. The GOI or DNA sequence of interest may be modified to be recognized by the viral integrase as needed. For example, the viral att sites can be added to the ends of the DNA sequence. C) Other reagents needed for polynucleotide transfection and/or introduction of protein into cells.

NUCLEIC ACID

0040The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably in this disclosure. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

GUIDE RNA

0041In aspects of the disclosure the terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and refer to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence. The term "guide sequence" refers to the about 20 bp (12-30 bp) sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms "guide" or "spacer". The term "tracr mate sequence" may also be used interchangeably with the term "direct repeat(s)".

WILD TYPE

0042As used herein the term "wild type" is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

VARIANT

0043As used herein the terms "variant" or "mutant" should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature. In relation to the genes, these terms indicate a number of changes in a gene that make it different from the wild-type gene including single nucleotide polymorphisms (SNPs), insertions, deletions, gene shifts among others.

ENGINEERED

0044The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of man-made technology. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

COMPLEMENTARY

0045"Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100%, or percentages in between over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

AMINO ACIDS

0046<ul id="ul0002" list-style="none" compact="compact"><li>Full Name, Three-Letter Code, One-Letter Code</li><li>Aspartic Acid Asp D</li><li>Glutamic Acid Glu E</li><li>Lysine Lys K</li><li>Arginine Arg R</li><li>Histidine His H</li><li>Tyrosine Tyr Y</li><li>Cysteine Cys C</li><li>Asparagine Asn N</li><li>Glutamine Gln Q</li><li>Serine Ser S</li><li>Threonine Thr T</li><li>Glycine Gly G</li><li>Alanine Ala A</li><li>Valine Val V</li><li>Leucine Leu L</li><li>Isoleucine Ile I</li><li>Methionine Met M</li><li>Proline Pro P</li><li>Phenylalanine Phe F</li><li>Tryptophan Trp W</li></ul>

0047The expression "amino acid" as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. "Standard amino acid" means any of the twenty standard L-amino acids commonly found in naturally occurring proteins/peptides. "Non-standard amino acid residue" means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present disclosure, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half-life without adversely affecting their activity. Additionally, a disulfide link may be present or absent in the peptides.

0048Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains; (2) side chains containing a hydroxyl (OH) group; (3) side chains containing sulfur atoms; (4) side chains containing an acidic or amide group; (5) side chains containing a basic group; (6) side chains containing an aromatic ring; and (7) proline, an imino acid in which the side chain is fused to the amino group.

0049As used herein, the term "conservative amino acid substitution" is defined herein as exchanges within one of the following five groups: <ol id="ol0001" compact="compact"><li>I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly;</li><li>II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin;</li><li>III. Polar, positively charged residues: His, Arg, Lys;</li><li>IV. Large, aliphatic, nonpolar residues: Met Leu, He, Val, Cys (Ile; autocorrect is not literate)</li><li>V. Large, aromatic residues: Phe, Tyr, Tip (Trp, likewise)</li></ol>

0050The present disclosure utilizes, unless otherwise provided, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See <nplcit id="ncit0012" npl-type="b"><text>Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989</text></nplcit>); <nplcit id="ncit0013" npl-type="b"><text>CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987</text></nplcit>)); the series <nplcit id="ncit0014" npl-type="b"><text>METHODS IN ENZYMOLOGY (Academic Press, Inc.</text></nplcit>): <nplcit id="ncit0015" npl-type="b"><text>PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995</text></nplcit>)), <nplcit id="ncit0016" npl-type="b"><text>Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL</text></nplcit>, and <nplcit id="ncit0017" npl-type="b"><text>ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987</text></nplcit>)).

VECTORS

0051Gene expression vectors (DNA-based or viral) will be used to express the fusion integrases in cells or tissues as well as to provide the DNA sequence (or gene) of interest with the appropriate sites needed for the integrase or recombinase to integrate that DNA (or gene) into the genome of the host species or cell. A number of gene expression vectors are known in the art. Vectors will be use for the gene of interest (or DNA sequence of interest). Vectors may be cut with a number of restriction enzymes known in the art.

CRISPR/CAS9

0052CRISPR/Cas9 is described in <patcit id="pcit0005" dnum="US8697359B"><text>US Patent 8697359</text></patcit>, <patcit id="pcit0006" dnum="US8889356B"><text>US 8889356</text></patcit> and <nplcit id="ncit0018" npl-type="s"><text>Ran et al (Nature Protocols, 2013, volume 8, pages 2281-2308</text></nplcit>). Cas9 protein utilizes RNA guides in order to bind specific sequences of DNA in a genome. The RNA guides (guide RNAs) may be designed to be from 10 to 40, from 12 to 35, from 15 to 30, or for example, from 18 to 22, or 20 nucleotides in length. See <nplcit id="ncit0019" npl-type="s"><text>Hsu et al, Nature Biotechnology, September 2013, volume 31, pages 827-832</text></nplcit>, which uses Cas9 from Streptococcus pyogenes. Another key Cas9 is from Staphylococcus Aureus (a smaller Cas9 than that of S pyogenes). The Cas9 protein utilizes guide RNAs to bind specific regions of a DNA sequence.

0053A catalytically inactive form of Cas9 is described in <nplcit id="ncit0020" npl-type="s"><text>Guilinger et al, Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification, Nature Biotechnology, April 25, 2014, volume 32, pages 577-582</text></nplcit>. Ciuilinger et al attached the catalytically inactive Cas9 to a Fok1 enzyme to achieve greater specificity in making cuts in genomic DNA. This catalytically inactive Cas9 allows for Cas9 to use RNA guides for binding of genomic DNA, while not being able to cut the DNA.

0054Cas9 is also available in its natural wt form, and also a human optimized codon form for better expression of Cas9 constructs in cells. (see <nplcit id="ncit0021" npl-type="s"><text>Mali et al, Science, 2013, volume 339, pages 823-826</text></nplcit>). Codon optimization of Cas9 may be conducted dependent on the species for its expression. Depending on whether one produces a protein form of the Integrase/Cas9 fusion protein (also known as ABBIE1) or a nucleotide expression vector form, the optimized or non-optimized (wt ) form may be used.

0055RNA guides toward a specific DNA sequence can be designed by various computer-based tools.

CRISPR/CPF1

0056Cpf1 is another protein, which uses a guide RNA in order to bind a specific sequence in genomic DNA. Cpf1 also cuts DNA making a staggered cut. Cpf1 may be made to be catalytically inactive for cutting ability.

OTHER CRISPR PROTEINS

0057These are proteins that utilize a guide RNA to target a specific DNA sequence and whether they have the ability to cut DNA or not. Some of these proteins may naturally have other enzymatic/catalytic functions.

TALEN

0058Transcription Activator-Like Effector Nucleases (TALENs) are fusion proteins with restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage and represent powerful tools for genome editing in situ. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left-TALEN and a right- TALEN, which references the handedness of DNA. See US 8, 440,432.

0059TAL effectors are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids. These two locations are highly variable (Repeat Variable Diresidues (RVD)) and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

0060The integrase or recombinase can be used to construct hybrid integrase or recombinase that are active in a yeast or cell assay. These reagents are also active in plant cells and in animal cells. TALEN studies used the wild-type Fokl cleavage domain, but some subsequent TALEN studies also used Fokl cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity. Both the number of amino acid residues between the TALEN DNA binding domain and the integrase or recombinase domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. The number of amino acid residues between the TALEN DNA binding domain and the integrase or recombinase domain may be modified by introduction of a spacer (distinct from the spacer sequence) between the plurality of TAL effector repeat sequences and the integrase or recombinase domain. The spacer sequence may be 6 to 102 or 9 to 30 nucleotides or 15 to 21 nucleotides. These spacers will usually not provide other activity to the hybrid protein besides providing a link between the DNA targeting protein (Cas9, TALE or zinc finger protein) and the integrase or recombinase. The amino acids for the spacers and for other uses in the instant disclosure are

0061The relationship between amino acid sequence and DNA recognition of the TALEN binding domain allows for designable proteins. In this case artificial gene synthesis is problematic because of improper annealing of the repetitive sequence found in the TALE binding domain. One solution to this is to use a publicly available software program named DNAWorks to find oligonucleotides suitable for assembly in a two step PCR; oligonucleotide assembly followed by whole gene amplification. A number of modular assembly methods for generating engineered TALE constructs have also been reported in the art.

0062Once the TALEN genes have been assembled together they are inserted into plasmids; the plasmids are then used to transfect the target cell where the gene products are expressed and enter the nucleus to access the genome. TALENs can be used to edit genomes by inducing double-strand breaks (DSB), which cells respond to with DNA repair, however, the instant disclosure seeks to use the power of viral integrases or bacterial or phage recombinases to insert DNA sequences of interest into targeted sites in the genome. See disclosure of <patcit id="pcit0007" dnum="WO2014134412A"><text>WO 2014134412</text></patcit> and <patcit id="pcit0008" dnum="US8748134B"><text>US Patent 8748134</text></patcit>.

ZINC FINGER PROTEINS

0063Zinc finger proteins for binding DNA and their design are described in <patcit id="pcit0009" dnum="US7928195B"><text>US 7928195</text></patcit>, <patcit id="pcit0010" dnum="US20090111188A"><text>US 2009/0111188</text></patcit>, and <patcit id="pcit0011" dnum="US7951925B"><text>US 7951925</text></patcit>. Zinc finger proteins utilize a number of linked zinc finger domains in a specified order to bind to a specific sequence of DNA. Zinc finger protein endonucleases have been well-established.

0064Zinc finger proteins (ZFPs) are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. A single zinc finger domain of this class of ZFPs is about 30 amino acids in length, and several structural studies have demonstrated that it contains a beta turn (containing two conserved cysteine residues) and an alpha helix (containing two conserved histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines. This class of ZFPs is also known as C2H2 ZFPs. Additional classes of ZFPs have also been suggested. See, e.g., <nplcit id="ncit0022" npl-type="s"><text>Jiang et al. (1996) J. Biol. Chem. 271:10723-10730</text></nplcit> for a discussion of Cys-Cys-His-Cys (C3H) ZFPs. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA recognition, but also in RNA binding and in protein-protein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.

0065Many zinc finger proteins have conserved cysteine and histidine residues that tetrahedrally-coordinate the single zinc atom in each finger domain. In particular, most ZFPs are characterized by finger components of the general sequence: -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His- (SEQ ID NO:49, in which X represents any amino acid (the C2H2 ZFPs). The zinc-coordinating sequences of this most widely represented class contain two cysteines and two histidines with particular spacings. The folded structure of each finger contains an antiparallel β-turn, a finger tip region and a short amphipathic α-helix. The metal coordinating ligands bind to the zinc ion and, in the case of zif268-type zinc fingers, the short amphipathic α-helix binds in the major groove of DNA. In addition, the structure of the zinc finger is stabilized by certain conserved hydrophobic amino acid residues (e.g., the residue directly preceding the first conserved Cys and the residue at position +4 of the helical segment of the finger) and by zinc coordination through the conserved cysteine and histidine residues.

OTHER DNA BINDING PROTEINS THAT MAY BIND SPECIFIC TARGET SEQUENCES IN GENOMIC DNA

0066The proteins include those unrelated to the zinc finger proteins, TALEN and CRISPR proteins that may bind to specific sequences in genomic DNA of various organisms. These may include transcription factors, transcriptional repressors, meganucleases, endonuclease DNA binding domains and others.

INTEGRASES

0067Integrases and endonuclease fusion proteins thereof are described in <patcit id="pcit0012" dnum="US20090011509A"><text>US 2009/0011509</text></patcit>. Integrases introduced are lentiviral integrase and HIV1 (human immunodeficiency virus 1) integrase. The instant disclosure fuses a catalytically inactive (or active) Cas9, TALE or Zinc finger protein to an integrase to target the integrase to a specific region of DNA in the genome that is chosen by the user.

0068The HIV-1 integrase, like other retroviral integrases, is able to recognize special features at the ends of the viral DNA located in the U3 and U5 regions of the long terminal repeats (LTRs) (Brown, 1997). The LTR termini are the only viral sequences thought to be required in cis for recognition by the integration machinery of retroviruses. Short imperfect inverted repeats are present at the outer edges of the LTRs in both murine and avian retroviruses (reviewed by Reicin et al., 1995). Along with the subterminal CA located at the outermost positions 3 and 4 in retroviral DNA ends (positions 1 and 2 being the 3' end processed nucleotides, these sequences are both necessary and sufficient for correct proviral integration in vitro and in vivo. Sequences internal to the CA dinucleotide appear to be important for optimal integrase activity (Brin &amp; Leis, 2002a; Brin &amp; Leis, 2002b; Brown, 1997). The terminal 15 bp of the HIV-1 LTRs have been shown to be crucial for correct 3' end processing and strand transfer reactions in vitro (Reicin et al., 1995; Brown, 1997). Longer substrates are used more efficiently than shorter ones by HIV-1 IN which indicates that binding interactions extend at least 14-21 bp inward from the viral DNA end. Brin and Leis (2002a) analysed the specific features of the HIV-1 LTRs and concluded that both the U3 and U5 LTR recognition sequences are required for IN-catalysed concerted DNA integration, even though the U5 LTRs are more efficient substrates for IN processing in vitro (Bushman &amp; Craigie, 1991; Sherman et al., 1992). The positions 17-20 of the IN recognition sequences are needed for a concerted DNA integration mechanism, but the HIV-1 IN tolerates considerable variation in both the U3 and U5 termini extending from the invariant subterminal CA dinucleotide (Brin &amp; Leis, 2002b). The instant disclosure includes a DNA vector that contains viral (retroviral or HIV) LTR regions at the 5' and 3' ends of a location to house the DNA sequence or gene of interest to be integrated into the genome. The LTR regions do not have to be the full length LTRs as long as they function to interact with the integrase for proper integration. The LTR regions may be modified to contain detectable (e.g. fluorescent), PCR detection, or selectable markers (e.g. antibiotic resistance). The vector is designed to be cut and linearized so that the LTR regions are at the 5' and 3' ends of the DNA fragment (via designed restriction sites to restriction endonuclease).

0069Integrases consist of three domains connected by flexible linkers. These domains are an N-terminal HH-CC zinc-binding domain, a catalytic core domain and a C-terminal DNA binding domain (<nplcit id="ncit0023" npl-type="s"><text>Lodi et al, Biochemistry, 1995, volume 34, pages 9826-9833</text></nplcit>). In some aspects of the disclosure the integrase bound to the Cas9 (or other DNA binding molecule) will not have the C-terminal binding domain. In one aspect of the disclosure, two different fusion proteins will be produced where one has catalytically inactive Cas9 (or TALE or zinc finger protein) fused with the N-terminal zinc binding domain of an integrase and the other has catalytically inactive Cas9 (or TALE or zinc finger protein) fused with the catalytic core domain of the integrase. The two different fusion proteins will be designed to bind to opposite strands of the genomic DNA as seen with TALE-Fok1 or Zinc finger-Fok1 systems. In this manner, when the N-terminal domain and the catalytic core come in contact, at the site on the genomic DNA, it will exhibit integrase activity. As full activity of integrase has also been observed to involve tetramers of integrase, fusion proteins may be designed with 1, 2, 3, 4 integrase proteins linked by flexible linkers that may be 1 to 20 amino acids in length or 4-12 amino acids in length.

RECOMBINASES

0070Recombinases including Cre, Flp, R, Dre, Kw, and Gin recombinase are described in <patcit id="pcit0013" dnum="US8816153B"><text>US 8816153</text></patcit> and <patcit id="pcit0014" dnum="US20040003420A"><text>US 2004/0003420</text></patcit>. Recombinases such as Cre recombinase use LoxP sites in order to excise a sequence from the genome. Recombinases can be modified to become constitutively active for their recombination activity and also become less site specific. Thus, it is possible to target such constitutively active recombinase proteins with no sequence specificity to specific sequences of DNA in a genome by incorporating them into fusion proteins of the instant disclosure. In this manner, the CRISPR/Cas9, TALE or zinc finger protein domain specifies the DNA sequence where the recombinase will contribute its recombination activity. Such recombinase proteins may be wild-type, constitutively active or dead for recombinase activity. A Cas9-recombinase such as Cas9-Gin or Cas9-Cre may be produced by use of a linker sequence or by direct fusion.

NUCLEAR LOCALIZATION SIGNAL SEQUENCE (NLS) FOR FUSION PROTEINS

0071The signal peptide domain (also referred to as "NLS") is, for example, derived from yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus E1a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins (see <nplcit id="ncit0024" npl-type="s"><text>Boulikas, Crit. Rev. Eucar. Gene Expression, 3, 193-227 (1993</text></nplcit>)), simian virus 40 ("SV40") T-antigen (<nplcit id="ncit0025" npl-type="s"><text>Kalderon et. al, Cell, 39, 499-509 (1984</text></nplcit>)) or other proteins with known nuclear localization. The NLS is, for example, derived from the SV40 T-antigen, but may be other NLS sequences known in the art. Tandem NLS sequences may be used.

LINKER REGIONS

0072The various linkers used between fusion proteins/peptides being synthesized will be composed of amino acids. At the DNA level , these are represented by 3 base pair (bp) codons as known in the genetic code. Linkers may be from 1 to 1000 amino acids in length and any integer in between. For example, linkers are from 1 to 200 amino acids in length or linkers are from 1 to 20 amino acids in length.

EXPRESSION VECTORS

0073Many nucleic acids may be introduced into cells to lead to expression of a gene. As used herein, the term nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double-stranded or single-stranded (i.e., a sense or an antisense single strand). Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5- bromo-2'-doxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-0-methyl or 2'-0-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, <nplcit id="ncit0026" npl-type="s"><text>Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3): 187</text></nplcit>; and <nplcit id="ncit0027" npl-type="s"><text>Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5</text></nplcit>. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. Nucleic acid sequences can be operably linked to a regulatory region such as a promoter. Regulatory regions can be from any species. As used herein, operably linked refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid. Any type of promoter can be operably linked to a nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, and promoters responsive or unresponsive to a particular stimulus (e.g., inducible promoters).

0074Additional regions that may be useful in nucleic acid constructs, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression can sometimes be obtained without such additional elements.

0075A nucleic acid construct may be used that encodes signal peptides or selectable markers. Signaling (marker) peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface). Non-limiting examples of such selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphtransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). These markers are useful for selecting stable transformants in culture. Other selectable markers include fluorescent polypeptides, such as green fluorescent protein, red fluorescent, or yellow fluorescent protein.

0076Nucleic acid constructs can be introduced into cells of any type using a variety of biological techniques known in the art. Non-limiting examples of these techniques would include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, or calcium phosphate precipitation, that are capable of delivering nucleic acids to cells. A system called Nucleofection.TM. may also be used.

0077Nucleic acids can be incorporated into vectors. A vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA. A vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus/phage DNA segment. Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence or mRNA, respectively.

0078Many different types of vectors are known in the art. For example, plasmids and viral vectors, including retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non- transcribed sequences. Such vectors include plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1 , SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (P-elements, Tol-2, Frog Prince, piggyBac or others).

0079Bacterial and viral genes and proteins for use in the disclosure are listed below in the section entitled "SEQUENCES OF THE DISCLOSURE". Other viral integrases, for example, those from mouse mammary tumor virus (MMTV) and adenovirus can also be used in the methods and compositions disclosed herein.

0080A pooled population of edited cells are considered a mixture of cells that have received a gene edit and cells that have not.

EXEMPLARY ABBIE1 IN VITRO ASSAY

0081<ol id="ol0002" compact="compact"><li>1) Incubate ABBIE1 protein with guide RNA;</li><li>2) Incubate ABBIE1/guide RNA with donor DNA having partial LTRs to form pre-initiation complex;</li><li>3) Incubate pre-initiation complex with plasmid containing gene to be edited (e.g. CXCR4); and</li><li>4) PCR and DNA sequencing confirmations for donor DNA integration.</li></ol>

0082Cas9protocols are described in, for example, Gagnon et al., 2014, http://labs.mcb.harvard.edu/schier/VertEmbryo/Cas9_Protocols.pdf.

0083Assays for integrase activity are described in, for example, <nplcit id="ncit0028" npl-type="s"><text>Merkel et al., Methods, 2009, volume 47, pages 243-248</text></nplcit>.

EXAMPLES

0084The following examples are intended to provide illustrations of the application of the present disclosure. The following examples are not intended to completely define or otherwise limit the scope of the disclosure. One of skill in the art will appreciate that many other methods known in the art may be substituted in lieu of the ones specifically described or referenced herein.

EXAMPLE 1: DNA VECTORS FOR EXPRESSION CAS9-INTEGRASE FUSION PROTEINS

0085The DNA sequence of catalytically inactive Cas9 is incorporated into an expression vector with a 12, 15, 18, 21, 24, 27 or 30 bp spacer (codes for 4, 5, 6, 7, 8, 9 or 10 amino acids as the linker between the Cas9 and the integrase) and the HIV1 integrase. In other experiments, recombinases of bacterial or phage origin are used rather than integrases. These include Hin recombinase (SEQ ID NO: 25) and Cre recombinase (SEQ ID NO: 26) with or without mutations that allow them to recombine DNA at any other sites. A His or cMyc tag (or other sequence useful for protein purification) may be included to isolate the fusion protein. The expression vector uses a promoter that will be activated in the cells that will be provided with the vector. The CMV (cytomegalovirus promoter) is commonly used for expression vectors for mammalian cells. The U6 promoter is also commonly used. A T7 promoter may be used for in vitro transcription in certain embodiments.

EXAMPLE 2: DNA VECTOR FOR EXPRESSION OF THE DNA SEQUENCE OF INTEREST (GENE OF INTEREST)

0086The DNA sequence of interest will be inserted into the appropriate expression vector and sites will be appropriately added to the DNA sequence of interest so the HIV1 integrase will recognize the sequences for integration into the genome. These sites are termed att sites (U5 and U3 att sites) (see <nplcit id="ncit0029" npl-type="s"><text>Masuda et al, Journal of Virology, 1998, volume 72, pages 8396-8402</text></nplcit>). Homology arms for the target site in the genome can be included in regions flanking the 5' and 3' ends of the DNA (gene) sequence of interest (see <nplcit id="ncit0030" npl-type="s"><text>Ishii et al, PLOS ONE, September 24, 2014, DOI: 10.1371/journal.pone.0108236</text></nplcit>). When using a recombinase, the integrase recognition sites may not be included. Markers, such as drug resistance markers (e.g. blasticidin or puromycin), will be included in order to check for insertion of the DNA sequence of interest and to help assay for random insertions in the genome. These resistance markers can be engineered in such a way to remove them from the targeted genome landing pad For example flanking the puromycin resistance gene with a LoxP sites and introducing exogenously expressed CRE would remove the internal sequence leaving a scar containing a LoxP site.

EXAMPLE 3: DNA VECTOR FOR REVERSE TRANSCRIPTASE EXPRESSION

0087A reverse transcriptase may also be co-expressed in such systems as the designed DNA sequence (Gene) of interest in the vector will become expressed as RNA and will have to be converted back to DNA for integration by the integrase enzyme. The reverse transcriptase may be viral in origin (e.g. a retrovirus such as HIV1). This may be incorporated within the same vector as the DNA sequence of interest.

EXAMPLE 4: CO-EXPRESSION OF DNA TARGETING-INTEGRASES (OR RECOMBINASES) WITH DNA SEQUENCE OF INTEREST

0088Cells were electroporated for the vectors described above along with the Cas9 RNA guides required for the target site in the genome. In some experiments, vectors were created that expressed all of the components (fusion Cas9/integrase (or recombinase), the Cas9 RNA guides, and the DNA sequence of interest with integrase recognition sites and with or without homology arms). A reverse transcriptase may also be co-expressed in such systems as the designed DNA sequence (Gene) of interest in the vector will become expressed as RNA and will have to be converted back to DNA for integration by the integrase enzyme. The reverse transcriptase may be viral in origin (e.g. a retrovirus such as HIV1). In other experiments, the DNA sequence of interest in linearized before introduction to the cell. The Cas9 RNA guide sequences and DNA sequence of interest had to be designed and inserted into the vector before use by standard molecular biology protocols.

EXAMPLE 5: TEST EXPERIMENTS AND ASSAYING FOR OFF-TARGET INSERTIONS

0089Cells missing expression of a particular gene, such as mouse embryonic fibroblasts from a knockout mouse model or cells genetically engineered to be knockouts for a given gene, are transfected or electroporated with the above vectors where the gene of interest is included. Chimeric primer sets designed to cover the inserted gene as well as flanking genomic sequence will be used to screen initial pools of edited cells. Limited dilution cloning (LDC) and or FACS analysis is then performed to ensure monoclonality. Next generation sequencing (NGS) or single nucleotide polymorphism (SNP) analysis is performed as a final quality control step to ensure isolated clones are homogenous for the designed edit. Other mechanisms for screening can include but are not limited to qRT-PCR and western blotting with appropriate antibodies. If the protein is associated with a certain phenotype of the cells, the cells may be examined for rescue of that phenotype. The genomes of the cells are assayed for the specificity of the DNA insertion and to find the relative number of off-target insertions, if any.

EXAMPLE 6: CAS9 LINKED INTEGRASE PROTEIN EXPRESSION AND ISOLATION

0090Vectors designed for gene expression in E coli or insect cells will be incorporated into E coli or insect cells and allowed to express for a given period of time. Several designs will be utilized to generate Cas9 (or inactive Cas9) linked integrase protein. The vectors will also incorporate a tag that is not limited to a His or cMyc tag for eventual isolation of the protein with high purity and yield. Preparation of the chimeric protein will include but are not limited to standard chromatography techniques. The protein may also be designed with one or more NLS (nuclear localization signal sequence) and/or a TAT sequence. The nuclear localization signal allows the protein to enter the nucleus. The TAT sequence allows for easier entry of a protein into a cell (it is a cell-penetrating peptide). Other cell penetrating peptides in the art may be considered. After sufficient time for expression has occurred, protein lysate will be collected from the cells and purified in the appropriate column depending on the tag used. The purified protein will then be placed in the appropriate buffering solution and stored at either -20 or -80 degrees C.

EXAMPLE 7: USING CAS9-INTEGRASE TO INCORPORATE STOP CODONS JUST UPSTREAM OF TRANSCRIPTION START SITE

0091The disclosure includes a method to create a knockout cell line or organism. The above system is used with the DNA sequence of interest being 1, 3, 6, 10, 15 or 20 consecutive stop codons to be placed just after the ATG start site for the target gene. This will create an effective gene knockout as transcription/translation will be stopped when reaching the immediate stop codon after the ATG start site. Additional stop codons will help prevent possible run through of the transcriptase (if transcriptase by-passes the first stop codon).

EXAMPLE 8: USING ABBIE1 (OR OTHER VARIATIONS HAVING OTHER SPECIFIC DNA BINDING DOMAINS) AS A PURIFIED PROTEIN TO EDIT THE GENOMES OF CELLS

0092Incubate Abbie1 isolated protein (other specific DNA sequence binding protein linked to retroviral integrase) with insertable/integratable DNA having viral LTR regions in a suitable buffer. (for formation of tetramer or other multimer depending on the instance). Alternatively, a premade composition of isolated Abbie1 protein with guide RNA may be combined with the insertable DNA sequence. Include guide RNA and incubate to incorporate guide RNA. Transfect or electroporate (or other technique of providing protein to cells) Abbie1/DNA preparation into cells. Allow time for genome/DNA editing to take place. Check for insertion of designed insertable DNA sequence into the specific site of the genomic DNA of the cell. Check for non-specific insertions by PCR and DNA sequencing.

0093As currently planned, the bacterial expression vector will be the pMAL-c5e, which is a discontinued product from NEB and one of the in-house cloning choices for Genscript. Codon-optimized Spy Cas9 is cloned with the his-tag and the TEV protease cleavage site in frame with the maltose-binding protein (MBP) tag. The ORF is under the inducible Tac promoter, and the vector also codes for the lac repressor (LacI) for tighter regulation. MBP will be used only as a stabilization tag and not a purification tag, for the amylose resin is quite expensive. The soluble expressed material will be purified over the Ni-affinity chromatography, then Cas9 is released by the TEV protease from MBP, purified by cation exchange chromatography, and polished by gel filtration.

EXAMPLE 9: DESIGN OF CONSTRUCTS FOR FUSION PROTEINS

0094Design sequence specific Zinc finger domain, TALE, or guide RNA for CRISPR based approach toward a target DNA sequence. Use on-line design software of choice.

0095Produce DNA construct with coding sequences for integrase, transposase or recombinase; a suitable amino acid linker; the appropriate zinc finger, TALE or CRISPR protein (e.g. Cas9, Cpf1); and an nuclear localization signal (or mitochondrial localization signal) to form the site specific fusion integrase protein. These are envisioned in multiple arrangements. A suitable tag may be included for protein isolation and purification if desired (e.g. maltose binding protein (MBP) or His tag).

0096DNA construct may utilize a mammalian cell promoter or a bacterial promoter common in the art (e.g. CMV, T7, etc.)

0097One may produce a recombinant fusion protein with E coli as the source. Isolate the protein by standard means in the art (e.g. MBP columns, nickel-sepharose columns, etc.).

0098Assemble the Donor-RNP complex (duplex the RNA oligos and mix with fusion protein of the invention (when fusion protein has an endonuclease inactive CRISPR related protein for its DNA binding ability, e.g. ABBIE1) - these steps of forming RNP are not necessary for Zinc finger domains and TALE. <ol id="ol0003" compact="compact"><li>1. Mix Donor DNA with appropriate LTR domains and insertable sequence, and fusion protein and incubate for 10 minutes. (alternatively add Donor DNA after the RNP complex formation)</li><li>2. Resuspend each RNA oligo (crRNA and tracrRNA) in Nuclease-Free IDTE Buffer. For example, use a final concentration of 100 µM.</li><li>3. Mix the two RNA oligos in equimolar concentrations in a sterile microcentrifuge tube. For example, create a final duplex concentration of 3 µM using the following table: Component Amount 100 µM crRNA 3 µL 100 µM tracrRNA 3 µL Nuclease-Free Duplex Buffer 94 µL Final volume 100 µL</li><li>4. Heat at 95° C for 5 min.</li><li>5. Remove from heat and allow to cool to room temperature (15-25°C) on your bench top.</li><li>6. If needed, dilute duplexed RNA to a working concentration (for example, 3 µM) in Nuclease-Free Duplex Buffer.</li><li>7. Dilute fusion protein to a working concentration (for example, 5 µM) in Working Buffer (20 mM HEPES, 150 mM KCI, 5% Glycerol, 1mM DTT, pH 7.5).</li><li>8. For each transfection, combine 1.5 pmol of duplexed RNA oligos (Step A5) with 1.5 pmol of fusion protein (Step A6) in Opti-MEM Media to a final volume of 12.5 µL.</li><li>9. Incubate at room temperature for 5 min to assemble the RNP complexes.</li></ol>

EXAMPLE 10: REVERSE TRANSFECT GRNA-FUSION PROTEIN IN A 96-WELL PLATE

00991. Incubate the following at room temperature for 20 min to form transfection complexes: Component Amount RNP (Step A8) 12.5 µL Lipofectamine^® RNAiMAX Transfection Reagent 1.2 µL Opti-MEM^® Media 11.3 µL Total volume 25.0 µL

01002. During incubation (Step B1), dilute cultured cells to 400,000 cells/mL using complete media without antibiotics.

01013. When incubation is complete, add 25 µL of transfection complexes (from Step B1) to a 96-well tissue culture plate.

01024. Add 125 µL of diluted cells (from Step B2) to the 96-well tissure culture plate (50,000 cells/well; final concentration of RNP will be 10 nM).

01035. Incubate the plate containing the transfection complexes and cells in a tissure culture incubator (37°C, 5% CO2 ) for 48 hr. To detect on-target mutations, use PCR with appropriate primers (primers within donor sequence and primers surrounding the target insertion site).

EXAMPLE 11: PROTOCOL FOR TESTING THE SPECIFICITY OF CRISPR/CAS9

0104Produce dCas9 (DNA cutting inactive Cas9) linked to biotin (dCas9-biotin). Cas9 (s pyogenes, s aureus, etc.). Biotinylation methods are described below.

0105Biotinylation method #1: engineer the avi-tag (~15 residues) at the N- or C-terminus, express and purify as the WT (un-tagged) protein. Use the E. coli biotin ligase (BirA) and biotin to biotinylate the avi-tagged Cas9. We use this scheme to biotinylate chemokines. I believe the IP on the avi-tag technology expired a few years ago.

0106Biotinylation method #2.1: biotin functionalized with succinimidyl-ester can be incorporated at surface-exposed lysines residues (no enzymatic reaction required). For proteins as big as Cas9, this can be a viable option.

0107Biotinylation method #2.2: along the same line, biotin-maleimide is commercially available, and they can be conjugated at surface-exposed cysteines (no enzyme).

0108Testing will be accomplished to characterize the biotinylated Cas9 does in terms of DNA-binding and cleavage.

0109Streptavidin-coated 96-well plates are commercially available, but may also be produced in-house.

0110Bind dCas9-biotin to plastic plates (96-well, 24-well, 384-well, etc.).

0111Provide designed guide RNAs to each well. Allow time for guide RNAs to interact with Cas9 protein.

0112Provide genomic DNA to each well or DNA with targeted sequence. Allow time for Cas9 binding to DNA.

0113Wash wells with appropriate buffer.

0114Provide an adapter (DNA oligomer). Allow time to bind.

0115Restriction-digest the genomic DNA to make it more tractable and easier to ligate the adapter.

0116Wash wells.

0117Perform DNA sequencing to identify sites of binding (on target vs. off target).

EXAMPLE 12: NRF2 EDITING VIA ABBIE 1

0118<figref idref="f0005"><b>FIG. 5</b></figref> shows Abbie1 Gene Editing Targeting Exon 2 of Nrf2 Using Guide NrF2-sgRNA2 and sgRNA3. PCR screen against exon 2 targeting Nrf2 locus for knock-out via Abbie1 Editing. Abbie1 transfection targeting exon 2 of Nrf2 using guide NrF2-sgRNA 2 and 3 showed integration of donor at targeted region. Unique bands are identified as 1-8.

0119<figref idref="f0006"><b>FIG. 6</b></figref> shows theoretical data generated by Abbie1 gene editing. Representation of DNA gel electrophoresis visualizing inserted donor DNA via the Abbie1 system to target genomic material using sgRNA 1-3. Black bands represent background product due to PCR methodology. Red bands represent unique products generated by amplifying insert and genetic material flanking the region of insert. Multiple bands represent possible multiple insertion in targeted region.

0120<figref idref="f0007"><b>FIG. 7</b></figref> shows Abbie1 Gene Editing Targeting Exon 2 of Nrf2 Using Guide Nrf2-sgRNA 3. PCR screen against exon 2 targeting Nrf2 locus for knock-out via Abbie1 Editing. Targeting exon 2 of Nrf2 using guide NrF2-sgRNA 3 suggested donor insertions as indicated by PCR primers designed to donor sequence and adjacent site to expected insertion. Unique bands are identified as 1-4

0121<figref idref="f0008"><b>FIG. 8</b></figref> shows Abbie1 Knock out of Nrf2 in pooled Hek293T Cells. (A)Western blot analysis using polyclonal antibody against 55kD isoform (Santa Cruz Bio) showing knock out of Nrf2 in pooled HEK293T poplulations. (B) GAPDH (Santa Cruz Bio) loading control.

0122<figref idref="f0009"><b>FIG. 9</b></figref> shows Abbie1 Knock out of Nrf2 in pooled Hek293T Cells. (A) Western blot analysis utilizing monoclonal antibody against Nrf2 (Abcam) showing knockout of Nrf2 pooled poplulations in HEK 293t cells. (B) GAPDH loading control. (C) Average of densitometric analysis showing decrease in expression ratios as compared to control.

0123Abbie1 treated cells generate a unique PCR band indicating integration of donor DNA. Phenotypic confirmation of knock out in a HEK293T pooled cell line was confirmed via western blot analysis probing for two isoforms with unique and different antibodies. ~80% knock out by integration was observed in pooled populations in under two weeks.

EXAMPLE 13: CXCR4 EDITING VIA ABBIE1

0124<figref idref="f0010"><b>FIG. 10</b></figref> shows Abbie1 Gene Editing Targeting CXCR4 Exon 2. PCR screen targeting exon 2 of CXCR4 edited via Abbie1. Four sets of primers were designed against the region of interest. Set number 2 and 4 appears to have generated unique bands suggesting integration of donor DNA at the region of interest.

EXAMPLE 14: TRANSFECTION FOR THE KNOCK-IN EXPERIMENT AT THE NRF2 LOCUS USING ABBIE1.

0125Note: 500 ng protein and 120ng sgRNA are used for a single reaction. The amount of DNA depends on the size of the donor constructs. Donor DNA (DNA with LTR sequences) may be incubated with ABBIE1 before, during, or after providing/transfecting/electroporating to the cells. All reactions are prepared in sterile biosafety cabinet.

0126Day 1: Human embryonic kidney (HEK 293T) Cells were seeded into 24-well culture plate (Corning) at 200,000 HEK293T cells (ATCC) per well in 500 µL DMEM (Gibco) supplemented with 10% fetal bovine serum (Omega Scientific). Cells were allowed to recover for 24 hours.

Day 2: ABBIE1 Preparation:

Tube 1:

0127Purified ABBIE1 protein (SEQ ID NO: 58) and donor DNA (SEQ ID NO: 101) in a reduced-serum transfection medium (OptiMEM, Life Technologies) at 1:1 molar ratio for 10 minutes at room temperature. Add the sgRNA to the 1.3-fold molar excess (approximately 120 ng) to the protein/DNA complex and continue the incubation for additional 10 minutes at room temperature. The volume of this mixture is 25 µL.

Tube 2:

01282µtL of transfection reagent (RNAiMAX, Life Technologies) was added to 23 µL of OptiMEM. And allowed to incubate for 10 minutes at room temperature.

0129Combined Tube 1 and Tube 2 (50ml final volume) and incubated for 15 minutes at room temperature.

0130Added the entire 50µL transfection mixture to the well.

0131Half of the pooled edited cells were harvested 48 hours after transfection for the verification of the genomic DNA editing in a pooled population. Verification of edited genome was performed by polymerase chain reaction (PCR). We performed PCR against the targeted region as described below (See PCR protocol) the remainder was seeded onto 6 cm culture dishes (Corning) and allowed to recover for 48 hours.

0132Day 5: Screening of phenotypic changes via western blotting.

0133Standard western blot analysis was performed for NrF2 isoforms using primary antibodies targeting 55kD isoform (Santa Cruz Biotechnology, sc-722) as well as 98kD isoform (Abcam, ab-62352). GAPDH (Santa Cruz Biotechnology, sc-51907)

EXAMPLE 15: PCR CONDITIONS FOR DETECTION OF GENE EDITING USING ABBIE1 FOR NRF2 AND CXCR4 LOCUS.

Accession number for human Nrf2

0134<ul id="ul0003" list-style="none" compact="compact"><li>Uniprot: Q16236</li><li>Ensembl gene ID: ENSG00000116044</li><li>Editing target sequences and PAMs for Nrf2 (exon 2): Used for sgRNA design 1-3.</li><li>GCGACGGAAAGAGTATGAGC TGG</li><li>TATTTGACTTCAGTCAGCGA CGG</li><li>TGGAGGCAAGATATAGATCT TGG</li></ul>

Primer Key for Detection of Integration at Nrf2 Target

0135<ul id="ul0004" list-style="none" compact="compact"><li>Primer Set 1: Primer 1:5'-GTGTTAATTTCAAACATCAGCAGC-3', Primer 2: 5'- GACAAGACATCCTTGATTTG-3'</li><li>Primer Set 2: Primer 1:5'-GAGGTTGACTGTGTAAATG-3', Primer 2: 5'-GATACCAGAGTCACACAACAG-3'</li><li>Primer Set 3: Primer 1: 5'-TCTACATTAATTCTCTTGTGC-3', Primer 2:5'-GATACCAGAGTCACACAACAG-3'</li></ul>

Accession number for human CXCR4

0136<ul id="ul0005" list-style="none" compact="compact"><li>Uniprot P61073</li><li>Ensembl gene ID: ENSG00000121966</li><li>Editing target sequence and PAM for CXCR4 (Exon 2): Used for sgRNA design1.</li><li>GGGCAATGGATTGGTCATCC TGG</li></ul>

Primer Key for Detection of Integration at CXCR4 Target

0137<ul id="ul0006" list-style="none" compact="compact"><li>Primer Set 1: Primer 1: 5'- TCTACATTAATTCTCTTGTGC-3', Primer 2: 5'-GACAAGACATCCTTGATTTG-3'</li><li>Primer Set 2: Primer 1: 5'- TCTACATTAATTCTCTTGTGC-3', Primer 2: 5'-GATACCAGAGTCACACAACAG -3'</li><li>Primer Set 3: Primer 1: 5'- GAGGTTGACTGTGTAAATG -3', Primer 2: 5'-GACAAGACATCCTTGATTTG-3'</li><li>Primer Set 4: Primer 1: 5'- GAGGTTGACTGTGTAAATG -3', Primer 2: 5'-GATACCAGAGTCACACAACAG -3'</li></ul>

PCR Cycling conditions used for detection of integrated donor DNA

0138*Note annealing temperatures will vary depending on primer sequence <tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39mm" /><colspec colnum="2" colname="col2" colwidth="22mm" /><colspec colnum="3" colname="col3" colwidth="16mm" /><tbody><row><entry>1. Initial denaturation:</entry><entry>4 min</entry><entry>94°C</entry></row><row><entry>2. denaturation:</entry><entry>30 sec</entry><entry>94°C</entry></row><row><entry>3. annealing:</entry><entry>30 sec</entry><entry>55°C</entry></row><row><entry>4. extension:</entry><entry>30 sec</entry><entry>72°C</entry></row><row><entry>5. go to step 2:</entry><entry>40 cycles</entry><entry /></row><row><entry>6. final extension:</entry><entry>4 min</entry><entry>72°C</entry></row><row><entry>7. final hold:</entry><entry>∞</entry><entry>4°C</entry></row></tbody></tgroup></table></tables>

Avi-tagged Cas9 for biotinylation

0139Sequence of the avi-tag used for Cas9 biotinylation <ul id="ul0007" list-style="none" compact="compact"><li><u>Amino acid seqeunce:</u><u>G G D</u> L E G S <u>G L N D I F E A Q K I E W H E *</u></li><li><u>Nucleic acid sequence:</u><img file="EP4600366A2_D0001.tif" /></li><li>First Underlined section = Cas9 C-terminus</li><li>Italicized section = restriction site/linker</li><li>Second underlined section = avi-tag (biotinylation site highlighted)</li></ul>

EXAMPLE 16: EXPRESSION PROTOCOL FOR ABBIE1 FUSION PROTEIN.

0140Transformation of expression construct containing full-length fusion protein (SEQ ID NO: 57).

0141Take competent E. coli cells from -80°C freezer.

0142Turn on water bath to 42°C.

0143Put competent cells in a 1.5 ml tube (Eppendorf or similar). For transforming a DNA construct, use 50 ul of competent cells.

0144Keep tubes on ice.

0145Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 min. to thaw competent cells.

0146Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds. Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.

0147Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37 oC. (Can incubate tubes for 30 minutes

0148Spread about 100 ul of the resulting culture on LB plates with appropriate antibiotic

0149Pick colonies about 12-16 hours later.

INNOCULATION AND EXPANSION

0150Innoculate a 1 liter flask containing LB and antibiotic

0151Allow bacterial culture to grow until 0.6 OD is achieved then induce with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a 1mM final concentration

0152Allow the culture to expand for 6-8 hours and centrifuge the suspended bacterial culture at a minimum of two thousand G force for 10 minutes.

0153Freeze pellet at -80 C for further processing at a later time

PROTEIN PREPARATION AND PURIFICATION

0154All steps are performed at room temperature.

0155Lyse the cells by 2 cycles of freeze-thaw in 20 mM Tris pH8.0, 300 mM NaCl, 0.1 mg/ml chicken egg white lysozyme. Centrifuge at 6,000 g for 15 minutes and retain the supernatant.

0156Load the supernatant onto a Ni-IDA agarose column equilibriated in 20 mM Tris pH8.0, 300 mM sodium chloride. Elute the protein with a 0-to-200 mM gradient of imidazole. Identify the fractions containing the fusion protein by a 7% SDS-PAGE.

0157Pool the fractions and dilute with 20 mM Tris pH8.0 so that the final NaCl concentration is 50 mM. Load onto a Q-sepharose column and elute with a 0-to-500 mM gradient of sodium chloride. Identify the fractions containing the fusion protein by a 7% SDS-PAGE.

0158Pool the fractions and dilute with 20 mM Tris pH8.0 so that the final NaCl concentration is 100 mM. Load onto an SP-sepharose column and elute with a 0-to-500 mM gradient of sodium chloride. Identify the fractions containing the fusion protein by a 7% SDS-PAGE.

0159Pool the fractions, measure the concentration by its UV absorbance at 280 nm, and concentrate by a centrifugal filter to the final concentration of 400 µg/ml. Add glycerol to the final concentration of 50%. Store at -20°C.

0160While certain embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

SEQUENCES OF THE DISCLOSURE

0161For each sequence provided below, the following information is provided: type of sequence (nucleic acid or amino acid), source (e.g. E. coli), length, and identification number (if available).

0162A first polynucleotide of the disclosure can encode, for example, a Cas9, Cpfl, TALE, or ZnFn protein. A second polynucleotide of the disclosure can encode, for example, an integrase, transposase, or recombinase. Listed below are exemplary first and second polynucleotide sequences and protein sequences, along with exemplary linker sequences, that can be used in the compositions (constructs, fusion proteins) and methods described herein. Other polynucleotide sequences, protein sequences, or linker sequences may be provided in the disclosure that are not listed in Table 1 below, but can be used in the compositions (constructs, fusion proteins) and methods described herein. For example, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58, and/or portions thereof.

0163A linker can be any length, for example, 3 to 300 nucleotides in length, 6 to 60 nucleotides in length, or any length that will allow the first and second polynucleotide to be fused. A polypeptide can be made by an organism, e.g. E. coli or be made synthetically, or a combination of both.

0164Exemplary nucleic acid sequences: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27-47, 49, 55, 56, 57, 62, 64, 66, 68, 70, 79, 82, and 83.

0165Exemplary amino acid sequences: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 48, 50, 52, 58, 63, 65, 67, 69, 71, 72-78, and 80. <tables id="tabl0002" num="0002"><table frame="all"><title>TABLE 1: FIRST PROTEIN, SECOND PROTEIN, OR LINKER</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="59mm" /><colspec colnum="3" colname="col3" colwidth="44mm" /><thead valign="top"><row><entry align="center">first polynucleotide or protein sequence SEQ ID NO:</entry><entry align="center">second polynucleotide or protein sequence SEQ ID NO:</entry><entry align="center">linker sequence SEQ ID NO: or sequence</entry></row></thead><tbody><row><entry>1-14, 27-46, 50, 52, 56, 57, 68, 69, 72-78, 86-92, 200-253</entry><entry>15-26, 47, 48, 55, 57, 62-67, 70, 71, 79, 80</entry><entry>51, 54, 61, GGS</entry></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="all"><title>TABLE 2: PARTIAL LIST OF SEQUENCES</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="47mm" /><colspec colnum="3" colname="col3" colwidth="25mm" /><colspec colnum="4" colname="col4" colwidth="29mm" /><colspec colnum="5" colname="col5" colwidth="37mm" /><thead valign="top"><row><entry><b>Gene (protein)</b></entry><entry><b>Bacteria/Virus</b></entry><entry><b>DNA sequence</b></entry><entry><b>Protein sequence</b></entry><entry><b>SEQ ID (DNA, protein)</b></entry></row></thead><tbody><row><entry><b>Cas9</b></entry><entry>S. thermophilus</entry><entry>HQ712120.1</entry><entry>Q03JI6.1</entry><entry>SEQ ID NOS: 1, 2</entry></row><row><entry /><entry>P. multocida</entry><entry /><entry>Q9CLT2.1</entry><entry>SEQ ID NOS: 3, 4</entry></row><row><entry /><entry>S. mutans</entry><entry /><entry>Q8DTE3.1</entry><entry>SEQ ID NOS: 5, 6</entry></row><row><entry /><entry>N. meningitides</entry><entry /><entry>C9X1G5.1</entry><entry>SEQ ID NOS: 7, 8</entry></row><row><entry /><entry>S. mitis</entry><entry /><entry>KJQ69483.1</entry><entry>SEQ ID NOS: 9, 10</entry></row><row><entry /><entry>S. macacae</entry><entry /><entry>EHJ52063.1</entry><entry>SEQ ID NOS: 11,12</entry></row><row><entry /><entry>Staphylococcus Aureus</entry><entry /><entry>KKJ92487.1</entry><entry>SEQ ID NOS: 49, 50</entry></row><row><entry /><entry>S. pyogenes</entry><entry /><entry>AFV37892.1</entry><entry>SEQ ID NOS: 13, 14</entry></row><row><entry><b>Integrase</b></entry><entry>HIV1</entry><entry /><entry>ABR68182.1</entry><entry>SEQ ID NOS: 15, 16</entry></row><row><entry /><entry>Simian T-lymphocyte virus</entry><entry /><entry>AAA47841.1</entry><entry>SEQ ID NOS: 17, 18</entry></row><row><entry /><entry>S. pneumonia</entry><entry /><entry>CBW38769.1</entry><entry>SEQ ID NOS: 19, 20</entry></row><row><entry /><entry>E. coli</entry><entry /><entry>CAA41325.1</entry><entry>SEQ ID NOS: 21, 22</entry></row><row><entry /><entry>Lentivirus</entry><entry /><entry /><entry>SEQ ID NOS: 47, 48</entry></row><row><entry><b>Recombinase</b></entry><entry>Thermoanaerobacterium phage</entry><entry /><entry>YP_006546326.1</entry><entry>SEQ ID NOS: 23 24</entry></row></tbody></tgroup></table></tables>

ADDITIONAL SEQUENCES

0166<ul id="ul0008" list-style="none" compact="compact"><li><b>SEQ ID NO: 1</b> NAME: S.thermophilus Csn1 cds HQ712120.1 SEQUENCE: <img file="EP4600366A2_D0002.tif" /><img file="EP4600366A2_D0003.tif" /><img file="EP4600366A2_D0004.tif" /></li><li><b>SEQ ID NO: 2</b> SEQUENCE: <img file="EP4600366A2_D0005.tif" /></li><li><b>SEQ ID NO: 3</b> NAME: P.multocida Cas9 SEQUENCE: <img file="EP4600366A2_D0006.tif" /><img file="EP4600366A2_D0007.tif" /><img file="EP4600366A2_D0008.tif" /></li><li><b>SEQ ID NO: 4</b> SEQUENCE: <img file="EP4600366A2_D0009.tif" /></li><li><b>SEQ ID NO: 5</b> NAME: S.mutans Cas9 SEQUENCE: <img file="EP4600366A2_D0010.tif" /><img file="EP4600366A2_D0011.tif" /><img file="EP4600366A2_D0012.tif" /><img file="EP4600366A2_D0013.tif" /></li><li><b>SEQ ID NO: 6</b> SEQUENCE: <img file="EP4600366A2_D0014.tif" /><img file="EP4600366A2_D0015.tif" /></li><li><b>SEQ ID NO: 7</b> NAME: N.meningitides Cas9 SEQUENCE: <img file="EP4600366A2_D0016.tif" /><img file="EP4600366A2_D0017.tif" /><img file="EP4600366A2_D0018.tif" /></li><li><b>SEQ ID NO: 8</b> SEQUENCE: <img file="EP4600366A2_D0019.tif" /></li><li>SEQ ID NO: 9 SEQUENCE: <img file="EP4600366A2_D0020.tif" /><img file="EP4600366A2_D0021.tif" /><img file="EP4600366A2_D0022.tif" /></li><li><b>SEQ ID NO: 10</b> NAME: gi|777888062|gb|KJQ69483.1| CRISPR-associated endonuclease Cas9 [Streptococcus mitis] SEQUENCE: <img file="EP4600366A2_D0023.tif" /><img file="EP4600366A2_D0024.tif" /></li><li><b>SEQ ID NO: 11</b> SEQUENCE: <img file="EP4600366A2_D0025.tif" /><img file="EP4600366A2_D0026.tif" /><img file="EP4600366A2_D0027.tif" /><img file="EP4600366A2_D0028.tif" /></li><li><b>SEQ ID NO: 12</b> NAME: gi|357584860|gb|EHJ52063.1| CRISPR-associated protein Cas9/Csn1, subtype II/NMEMI [Streptococcus macacae NCTC 11558] SEQUENCE: <img file="EP4600366A2_D0029.tif" /><img file="EP4600366A2_D0030.tif" /></li><li><b>SEQ ID NO: 13</b> SEQUENCE: <img file="EP4600366A2_D0031.tif" /><img file="EP4600366A2_D0032.tif" /><img file="EP4600366A2_D0033.tif" /></li><li><b>SEQ ID NO: 14</b> NAME: gi|409693032|gb|AFV37892.1| CRISPR-associated protein, Csn1 family [Streptococcus pyogenes A20] SEQUENCE: <img file="EP4600366A2_D0034.tif" /><img file="EP4600366A2_D0035.tif" /></li><li><b>SEQ ID NO: 15</b> NAME: gi|150381361|gb|EF472760.1| HIV-1 clone 39B from USA integrase (pol) gene, partial cds SEQUENCE: <img file="EP4600366A2_D0036.tif" /><img file="EP4600366A2_D0037.tif" /></li><li><b>SEQ ID NO: 16</b> NAME: gi|150381362|gb|ABR68182.1| integrase, partial [Human immunodeficiency virus 1] SEQUENCE: <img file="EP4600366A2_D0038.tif" /></li><li><b>SEQ ID NO: 17</b> NAME: gi|459980|gb|L20651.1|STLKIAPOL Simian T-cell lymphotropic virus type I integrase (pol) gene, partial cds SEQUENCE: <img file="EP4600366A2_D0039.tif" /></li><li><b>SEQ ID NO: 18</b> NAME: gi|459981|gb|AAA47841.1| integrase, partial [Simian T-lymphotropic virus 1] SEQUENCE: LVERSNGILKTLLYKYFTDKPDLPMDNALSIALWTINHLNVLTHCH</li><li><b>SEQ ID NO: 19</b> NAME: gi|321156784:1-1509 Streptococcus pneumoniae integrative and conjugative element ICESpn11930, strain 11930 SEQUENCE: <img file="EP4600366A2_D0040.tif" /><img file="EP4600366A2_D0041.tif" /></li><li><b>SEQ ID NO: 20</b> NAME: gi|321156785|emb|CBW38769.1| Integrase [Streptococcus pneumoniae] SEQUENCE: <img file="EP4600366A2_D0042.tif" /><img file="EP4600366A2_D0043.tif" /></li><li><b>SEQ ID NO: 21</b> NAME: gi|43090:1-436 E.coli (Tn5086) dhfrVII gene for dihydrofolate reductase type VII and sulI gene, 5' end (integrase) SEQUENCE: <img file="EP4600366A2_D0044.tif" /></li><li><b>SEQ ID NO: 22</b> NAME: gi|43091|emb|CAA41325.1| integrase, partial (plasmid) [Escherichia coli] SEQUENCE: <img file="EP4600366A2_D0045.tif" /></li><li><b>SEQ ID NO: 23</b> >gi|397912605:40372-41898 Thermoanaerobacterium phage THSA-485A, complete genome - recombinase <img file="EP4600366A2_D0046.tif" /><img file="EP4600366A2_D0047.tif" /></li><li><b>SEQ ID NO: 24</b> >gi|397912662|ref|YP_006546326.1| Recombinase [Thermoanaerobacterium phage THSA-485A] <img file="EP4600366A2_D0048.tif" /><img file="EP4600366A2_D0049.tif" /></li><li><b>SEQ ID NO: 25</b> Gin recombinase >gi|657193240|sp|Q38199.2|GIN_BPD10 RecName: Full=Serine recombinase gin; AltName: Full=G-segment invertase; Short=Gin <img file="EP4600366A2_D0050.tif" /></li><li><b>SEQ ID NO: 26</b> Cre recombinase >gi|375331813|dbj|BAL61207.1| Cre recombinase [Cre-expression vector pHVX2-cre] <img file="EP4600366A2_D0051.tif" /></li><li><b><u>SEQ ID NOS: 27-46</u></b> These are exemplary sequences of polynucleotides encoding the TALE repeat modules for use in linking to integrases or recombinases as described in this invention.</li><li><b>SEQ ID NO: 27</b> NAME: NI SEQUENCE: <img file="EP4600366A2_D0052.tif" /></li><li><b>SEQ ID NO: 28</b> NAME: NG SEQUENCE: <img file="EP4600366A2_D0053.tif" /></li><li><b>SEQ ID NO: 29</b> NAME: HD SEQUENCE: <img file="EP4600366A2_D0054.tif" /></li><li><b>SEQ ID NO: 30</b> NAME: NN SEQUENCE: <img file="EP4600366A2_D0055.tif" /></li><li><b>SEQ ID NO: 31</b> NAME: NI-NI <img file="EP4600366A2_D0056.tif" /></li><li><b>SEQ ID NO: 32</b> NAME: NI-NG SEQUENCE: <img file="EP4600366A2_D0057.tif" /></li><li><b>SEQ ID NO: 33</b> NAME: NI-HD SEQUENCE: <img file="EP4600366A2_D0058.tif" /></li><li><b>SEQ ID NO: 34</b> NAME: NI-NN SEQUENCE: <img file="EP4600366A2_D0059.tif" /></li><li><b>SEQ ID NO: 35</b> NAME: NG-NI SEQUENCE: <img file="EP4600366A2_D0060.tif" /></li><li><b>SEQ ID NO: 36</b> NAME: NG-NG SEQUENCE: <img file="EP4600366A2_D0061.tif" /></li><li><b>SEQ ID NO: 37</b> NAME: NG-HD SEQUENCE: <img file="EP4600366A2_D0062.tif" /></li><li><b>SEQ ID NO: 38</b> NAME: NG-NN SEQUENCE: <img file="EP4600366A2_D0063.tif" /></li><li><b>SEQ ID NO: 39</b> NAME: HD-NI SEQUENCE: <img file="EP4600366A2_D0064.tif" /></li><li><b>SEQ ID NO: 40</b> NAME: HD-NG SEQUENCE: <img file="EP4600366A2_D0065.tif" /></li><li><b>SEQ ID NO: 41</b> NAME: HD-HD SEQUENCE: <img file="EP4600366A2_D0066.tif" /></li><li><b>SEQ ID NO: 42</b> NAME: HD-NN SEQUENCE: <img file="EP4600366A2_D0067.tif" /></li><li><b>SEQ ID NO: 43</b> NAME: NN-NI SEQUENCE: <img file="EP4600366A2_D0068.tif" /></li><li><b>SEQ ID NO: 44</b> NAME: NN-NG SEQUENCE: <img file="EP4600366A2_D0069.tif" /></li><li><b>SEQ ID NO: 45</b> NAME: NN-HD SEQUENCE: <img file="EP4600366A2_D0070.tif" /></li><li><b>SEQ ID NO: 46</b> NAME: NN-NN SEQUENCE: <img file="EP4600366A2_D0071.tif" /></li><li><b>SEQ ID NO: 47</b> NAME: gi|71796612|gb|DQ084353.1| Ovine lentivirus isolate Ov10 integrase (pol) gene, partial cds SEQUENCE: <img file="EP4600366A2_D0072.tif" /><img file="EP4600366A2_D0073.tif" /></li><li><b>SEQ ID NO: 48</b> NAME: gi|71796613|gb|AAZ41325.1| integrase, partial [Ovine lentivirus] SEQUENCE: <img file="EP4600366A2_D0074.tif" /></li><li><b>SEQ ID NO: 49</b> >gb|AYLT01000127.1|:11804-12046 Staphylococcus aureus subsp. aureus SK1585 contig000127, whole genome shotgun sequence <img file="EP4600366A2_D0075.tif" /></li><li><b>SEQ ID NO: 50</b> >gi|669035130|gb|KFD30483.1| hypothetical protein D484_02234 [Staphylococcus aureus subsp. aureus SK1585] - s aureus cas9 <img file="EP4600366A2_D0076.tif" /></li><li><b>SEQ ID NO: 51</b> NAME: dna of linker2 SEQUENCE: agcggcagcgaaaccccgggcaccagcgaaagcgcgaccccggaaagc</li><li><b>SEQ ID NO: 52</b> NAME: dCas9 protein SEQUENCE: <img file="EP4600366A2_D0077.tif" /><img file="EP4600366A2_D0078.tif" /></li><li><b>SEQ ID NO: 53</b> NAME: NLS nucleotide with ATG SEQUENCE: <img file="EP4600366A2_D0079.tif" /></li><li><b>SEQ ID NO: 54</b> NAME: GGS linker nucleotide SEQUENCE: GGGGGAAGT</li><li><b>SEQ ID NO: 55</b> NAME: Synthetic integrase SEQUENCE: <img file="EP4600366A2_D0080.tif" /><img file="EP4600366A2_D0081.tif" /></li><li><b>SEQ ID NO: 56</b> NAME: dCas9 nucleotide with ATG SEQUENCE: <img file="EP4600366A2_D0082.tif" /><img file="EP4600366A2_D0083.tif" /><img file="EP4600366A2_D0084.tif" /><img file="EP4600366A2_D0085.tif" /><img file="EP4600366A2_D0086.tif" /><img file="EP4600366A2_D0087.tif" /></li><li><b>SEQ ID NO: 57</b> NAME: ABBIE1 (NLS-linker1-Integrase-linker2-dCas9) -DNA sequence SEQUENCE: <img file="EP4600366A2_D0088.tif" /><img file="EP4600366A2_D0089.tif" /><img file="EP4600366A2_D0090.tif" /><img file="EP4600366A2_D0091.tif" /><img file="EP4600366A2_D0092.tif" /><img file="EP4600366A2_D0093.tif" /></li><li><b>SEQ ID NO: 58</b> NAME: Translation of ABBIE1 (A Binding Based Integrase Editor) SEQUENCE: <img file="EP4600366A2_D0094.tif" /><img file="EP4600366A2_D0095.tif" /><img file="EP4600366A2_D0096.tif" /> For donor DNA (att sites of LTR regions for integrase recognition).</li><li><b>SEQ ID NO: 59</b> NAME: U3att SEQUENCE: ACTGGAAGGGCTAATTCACTCCCAAAGAA</li><li><b>SEQ ID NO: 60</b> NAME: U5att SEQUENCE: GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT NLS-linker1-Integrase-linker2-dCas9, or Integrase-linker1-NLS-linker2-dCas9 or Integrase-linker2-dCas9-linker1-NLS or Integrase-linker2-dCas9-NLS Linker 1 = GGS</li><li><b>SEQ ID NO: 61</b> NAME: Linker 2 SEQUENCE: SGSETPGTSESATPES</li><li><b>SEQ ID NO: 62</b> NAME: MMTV integrase cDNA, gb|AF071010.1|:16-1113 Mouse mammary tumor virus putative integrase, env polyprotein, and superantigen mRNA, complete cds SEQUENCE: <img file="EP4600366A2_D0097.tif" /><img file="EP4600366A2_D0098.tif" /></li><li><b>SEQ ID NO: 63</b> NAME: gi|3273866|gb|AAC24859.1| putative integrase [Mouse mammary tumor virus] SEQUENCE: <img file="EP4600366A2_D0099.tif" /><img file="EP4600366A2_D0100.tif" /></li><li><b>SEQ ID NO: 64</b> NAME: gb|AXUN02000059.1|:5116-8850 Youngiibacter fragilis 232.1 contig_151, whole genome shotgun sequence - recombinase SEQUENCE: <img file="EP4600366A2_D0101.tif" /><img file="EP4600366A2_D0102.tif" /><img file="EP4600366A2_D0103.tif" /><img file="EP4600366A2_D0104.tif" /></li><li><b>SEQ ID NO: 65</b> NAME: gi|564135645|gb|ETA81829.1| recombinase [Youngiibacter fragilis 232.1] SEQUENCE: <img file="EP4600366A2_D0105.tif" /><img file="EP4600366A2_D0106.tif" /></li><li><b>SEQ ID NO: 66</b> NAME: gi|571264543:16423-16770 Clostridium difficile transposon Tn6218, strain Ox42 Transposase SEQUENCE: <img file="EP4600366A2_D0107.tif" /></li><li><b>SEQ ID NO: 67</b> NAME: gi|571264559|emb|CDF47133.1| transposase [Peptoclostridium difficile] SEQUENCE: <img file="EP4600366A2_D0108.tif" /></li><li><b>SEQ ID NO: 68</b> NAME: gb|CP009444.1|:1317724-1320543 Francisella philomiragia strain GA01-2801, complete genome Cpf1 SEQUENCE: <img file="EP4600366A2_D0109.tif" /><img file="EP4600366A2_D0110.tif" /><img file="EP4600366A2_D0111.tif" /><img file="EP4600366A2_D0112.tif" /></li><li><b>SEQ ID NO: 69</b> NAME: gi|754264888|gb|AJI57252.1| CRISPR-associated protein Cpfl, subtype PREFRAN [Francisella philomiragia] SEQUENCE: <img file="EP4600366A2_D0113.tif" /><img file="EP4600366A2_D0114.tif" /></li><li><b>SEQ ID NO: 70</b> NAME: gi|438609|gb|L21188.1|HIV1NY5A Human immunodeficiency virus type 1 integrase gene, 3' end SEQUENCE: <img file="EP4600366A2_D0115.tif" /><img file="EP4600366A2_D0116.tif" /></li><li><b>SEQ ID NO: 71</b> NAME: gi|438610|gb|AAC37875.1| integrase, partial [Human immunodeficiency virus 1] SEQUENCE: <img file="EP4600366A2_D0117.tif" /></li><li><b>SEQ ID NO: 72</b> NAME: gi|545612232|ref|WP_021736722.1| type V CRISPR-associated protein Cpfl [Acidaminococcus sp. BV3L6] SEQUENCE: <img file="EP4600366A2_D0118.tif" /><img file="EP4600366A2_D0119.tif" /></li><li><b>SEQ ID NO: 73</b> NAME: gi|769142322|ref|WP_044919442.1| type V CRISPR-associated protein Cpfl [Lachnospiraceae bacterium MA2020] SEQUENCE: <img file="EP4600366A2_D0120.tif" /><img file="EP4600366A2_D0121.tif" /></li><li><b>SEQ ID NO: 74</b> NAME: gi|489130501|ref|WP_003040289. 11 type V CRISPR-associated protein Cpfl [Francisella tularensis] SEQUENCE: <img file="EP4600366A2_D0122.tif" /><img file="EP4600366A2_D0123.tif" /></li><li><b>SEQ ID NO: 75</b> NAME: gi|502240446|ref|WP_012739647.1| type V CRISPR-associated protein Cpfl [[Eubacterium] eligens] SEQUENCE: <img file="EP4600366A2_D0124.tif" /><img file="EP4600366A2_D0125.tif" /></li><li><b>SEQ ID NO: 76</b> NAME: gi|537834683|ref|WP_020988726.1| type V CRISPR-associated protein Cpfl [Leptospira inadai] SEQUENCE: <img file="EP4600366A2_D0126.tif" /><img file="EP4600366A2_D0127.tif" /></li><li><b>SEQ ID NO: 77</b> NAME: gi|739008549|ref|WP_036890108.1| type V CRISPR-associated protein Cpfl [Porphyromonas crevioricanis] SEQUENCE: <img file="EP4600366A2_D0128.tif" /><img file="EP4600366A2_D0129.tif" /></li><li><b>SEQ ID NO: 78</b> NAME: gi|517171043|ref|WP_018359861.1| type V CRISPR-associated protein Cpfl [Porphyromonas macacae] SEQUENCE: <img file="EP4600366A2_D0130.tif" /><img file="EP4600366A2_D0131.tif" /></li><li><b>SEQ ID NO: 79</b> NAME: Integrase protein sequence found on the Uniprot site. DNA sequence was obtained from GenBank. SEQUENCE: <img file="EP4600366A2_D0132.tif" /><img file="EP4600366A2_D0133.tif" /></li><li><b>SEQ ID NO: 80</b> NAME: sp|P04585|1148-1435 SEQUENCE: <img file="EP4600366A2_D0134.tif" /></li><li><b>SEQ ID NO: 81</b> a protein domain that characterizes zinc finger proteins CX(2-4)CX(12)HX(3-5)H (X(2-4) means XX or XXX or XXXX for example)</li><li><b>SEQ ID NO: 82</b> >gi|1616606|emb|X97044.1| Mouse mammary tumor virus 5' LTR DNA <img file="EP4600366A2_D0135.tif" /><img file="EP4600366A2_D0136.tif" /><img file="EP4600366A2_D0137.tif" /></li><li><b>SEQ ID NO: 83</b> >gi|1403387|emb|X98457.1| Mouse mammary tumor virus 3' LTR <img file="EP4600366A2_D0138.tif" /><img file="EP4600366A2_D0139.tif" /></li><li><b>SEQ ID NO: 84</b> >gi|119662099|emb|AM076881.1| Human immunodeficiency virus 1 proviral 5' LTR, TAR element and U3, U5 and R repeat regions, clone PG232.14 <img file="EP4600366A2_D0140.tif" /></li><li><b>SEQ ID NO: 85</b> >gi|1072081|gb|U37267.1|HIV1U37267 Human immunodeficiency virus type 1 3' LTR region <img file="EP4600366A2_D0141.tif" /></li><li><b>THERE ARE NO SEQ ID NOS: 86-99</b></li><li><b>SEQ ID NO: 100</b> Oligo for insertion of neo into a cell's genome (using full sequences of 5' and 3' HIV LTRs <img file="EP4600366A2_D0142.tif" /><img file="EP4600366A2_D0143.tif" /> First 5'LTR is <u>underlined</u>, plain text is neo, and 3'LTR is <b>bolded</b> (1179 bp)</li><li><b>SEQ ID NO: 101</b> An abbreviated version of 5'LTR and 3'LTR with neo sequence within (224 bp) First 5'LTR is <u>underlined</u>, plain text is neo, and 3'LTR is <b>bolded</b><img file="EP4600366A2_D0144.tif" /><img file="EP4600366A2_D0145.tif" /></li><li><b>Regarding SEQ ID NO: 72</b><ul id="ul0009" list-style="none" compact="compact"><li>Genbank Protein ID: WP_021736722.1</li><li>NCBI protein GI from NR Database or local GI (for proteins originated from WGS database): 545612232</li><li>Contig ID in WGS database: AWUR01000016.1</li><li>Contig description: Acidaminococcus sp. BV3L6 contig00028, whole genome shotgun sequence</li><li>Protein completeness: Complete</li><li>Proteins analyzed experimentally: 8</li><li>Non-redundant set: nr</li><li>Organism: Acidaminococcus_sp_BV3L6</li><li>Taxonomy: Bacteria,Firmicutes,Negativicutes,Selenomonadales,Acidaminococcaceae,Acidaminococ cus,Acidaminococcus sp. BV3L6</li></ul></li><li><b>Regarding SEQ ID NO: 73</b><ul id="ul0010" list-style="none" compact="compact"><li>Genbank Protein ID: WP_044919442.1</li><li>NCBI protein GI from NR Database or local GI (for proteins originated from WGS database): 769142322</li><li>Contig ID in WGS database: JQKK01000008.1</li><li>Contig description: Lachnospiraceae bacterium MA2020</li><li>T348DRAFT_scaffold00007.7_C, whole genome shotgun sequence</li><li>Protein completeness: Complete</li><li>Proteins analyzed experimentally: 9</li><li>Non-redundant set: nr</li><li>Organism: Lachnospiraceae_bacterium_MA2020</li><li>Taxonomy: Bacteria,Firmicutes,Clostridia,Clostridiales,Lachnospiraceae,unclassified Lachnospiraceae,Lachnospiraceae bacterium MA2020</li></ul></li></ul>

0167<b>Additional nucleic acid sequences and protein sequences that can be used in the disclosed compositions and methods - CPF 1 alignment.</b> SEQ ID NOS: 86-92; in order from the top to the bottom of the chart. <img file="EP4600366A2_D0146.tif" /><img file="EP4600366A2_D0147.tif" /><img file="EP4600366A2_D0148.tif" /><img file="EP4600366A2_D0149.tif" />

0168<b>Additional nucleic acid sequences and protein sequences that can be used in the disclosed compositions and methods - Cfp1 human cleaving proteins alignment.</b> SEQ ID NO: 86 (first row) and SEQ ID NO: 90 (second row). <img file="EP4600366A2_D0150.tif" /><img file="EP4600366A2_D0151.tif" />

0169<b>Additional nucleic acid sequences and protein sequences that can be used in the disclosed compositions and methods.</b> Table taken from <nplcit id="ncit0031" npl-type="s"><text>Haft, D., et al. PLoS Computational Biology, November 2005, Vol. 1, Issue 6, pp. 474-483</text></nplcit>. SEQ ID NOS: 200-253; in order from the top to the bottom of the chart. <tables id="tabl0004" num="0004"><img file="EP4600366A2_D0152.tif" /></tables><tables id="tabl0005" num="0005"><img file="EP4600366A2_D0153.tif" /></tables>

Editing target sequences and PAMs for Nrf2 (exon 2): Used for sgRNA design 1-3

0170<ul id="ul0011" list-style="none" compact="compact"><li><b>SEQ ID NO: 254</b> GCGACGGAAAGAGTATGAGC TGG</li><li><b>SEQ ID NO: 255</b> TATTTGACTTCAGTCAGCGA CGG</li><li><b>SEQ ID NO: 256</b> TGGAGGCAAGATATAGATCT TGG</li></ul>

Primer Key for Detection of Integration at Nrf2 Target

0171<ul id="ul0012" list-style="none" compact="compact"><li>Primer Set 1: <ul id="ul0013" list-style="none" compact="compact"><li><b>SEQ ID NO: 257</b> Primer 1:5'-GTGTTAATTTCAAACATCAGCAGC-3',</li><li><b>SEQ ID NO: 258</b> Primer 2: 5'- GACAAGACATCCTTGATTTG-3'</li></ul></li><li>Primer Set 2: <ul id="ul0014" list-style="none" compact="compact"><li><b>SEQ ID NO: 259</b> Primer 1:5'-GAGGTTGACTGTGTAAATG-3',</li><li><b>SEQ ID NO: 260</b> Primer 2: 5'- GATACCAGAGTCACACAACAG-3'</li></ul></li><li>Primer Set 3: <ul id="ul0015" list-style="none" compact="compact"><li><b>SEQ ID NO: 261</b> Primer 1: 5'-TCTACATTAATTCTCTTGTGC-3',</li><li><b>SEQ ID NO: 262</b> Primer 2:5'- GATACCAGAGTCACACAACAG-3'</li></ul></li></ul>

Accession number for human CXCR4

0172<ul id="ul0016" list-style="none" compact="compact"><li>Uniprot P61073</li><li>Ensembl gene ID: ENSG00000121966</li><li>Editing target sequence and PAM for CXCR4 (Exon 2): Used for sgRNA design1</li><li><b>SEQ ID NO: 263</b> GGGCAATGGATTGGTCATCC TGG</li><li>Primer Key for Detection of Integration at CXCR4 Target</li><li>Primer Set 1: <b>SEQ ID NO: 264</b></li><li>Primer 1: 5'- TCTACATTAATTCTCTTGTGC-3', <b>SEQ ID NO: 265</b></li><li>Primer 2: 5'- GACAAGACATCCTTGATTTG-3'</li><li>Primer Set 2: <ul id="ul0017" list-style="none" compact="compact"><li><b>SEQ ID NO: 266</b> Primer 1: 5'- TCTACATTAATTCTCTTGTGC-3',</li><li><b>SEQ ID NO: 267</b> Primer 2: 5'- GATACCAGAGTCACACAACAG -3'</li></ul></li><li>Primer Set 3: <ul id="ul0018" list-style="none" compact="compact"><li><b>SEQ ID NO: 268</b> Primer 1: 5'- GAGGTTGACTGTGTAAATG -3',</li><li><b>SEQ ID NO: 269</b> Primer 2: 5'- GACAAGACATCCTTGATTTG-3'</li></ul></li><li>Primer Set 4: <ul id="ul0019" list-style="none" compact="compact"><li><b>SEQ ID NO: 270</b> Primer 1: 5'- GAGGTTGACTGTGTAAATG -3',</li><li><b>SEQ ID NO: 271</b> Primer 2: 5'- GATACCAGAGTCACACAACAG -3'</li></ul></li></ul>

Avi-tagged Cas9 for biotinylation

0173Sequence of the avi-tag used for Cas9 biotinylation <ul id="ul0020" list-style="none" compact="compact"><li>Amino acid seqeunce: <b>SEQ ID NO: 272</b> G G D L E G S G L N D I F E A Q K I E W H E*</li><li>Nucleic acid sequence: <b>SEQ ID NO: 273</b><img file="EP4600366A2_D0154.tif" /></li></ul>

<u>ASPECTS OF THE INVENTION</u>

0174<ol id="ol0004"><li>1. A nucleic acid construct comprising in operable linkage: <ol id="ol0005" compact="compact"><li>a) a first polynucleotide sequence encoding a Cas9, an inactive Cas9, or a Cpfl, or a portion thereof:</li><li>b) a second polynucleotide sequence encoding an integrase, a recombinase, or a transposase, or a portion thereof; and</li><li>c) a third polynucleotide sequence encoding a nucleic acid linker;</li></ol> wherein the first polynucleotide sequence comprises a 5' and a 3' end and the second polynucleotide sequence comprises a 5' and a 3' end, and the 3' end of the first polynucleotide is connected to the 5' end of the second polynucleotide by the nucleic acid linker, and the first and second polynucleotide are able to be expressed as a fusion protein in a cell or an organism.</li><li>2. The nucleic acid construct of aspect 1, wherein the first polynucleotide sequence comprises any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 27-46, 49, 56, or 68, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.</li><li>3. The nucleic acid construct of aspect 1, wherein the Cas9, an inactive Cas9, or a Cpf1comprises any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 50, 52, 69, 72-78, or 86-92, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.</li><li>4. The nucleic acid construct of aspect 1, wherein the second polynucleotide sequence comprises any one of SEQ ID NOS: 15, 17, 19, 21, 23, 47, 55, 62, 64, 66, 70, or 79, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.</li><li>5. The nucleic acid construct of aspect 1, wherein the integrase, recombinase, or transposase comprises any one of SEQ ID NOS: 16, 18, 20, 22, 24, 25, 26, 48, 63, 65, 67, 71, or 80, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.</li><li>6. An organism comprising the nucleic acid construct of aspect 1.</li><li>7. An organism comprising the fusion protein of aspect 1 wherein the organism has a modified genome.</li><li>8. An organism comprising: <ol id="ol0006" compact="compact"><li>a) a first polynucleotide sequence encoding a Cas9, an inactive Cas9, or a Cpfl, or a portion thereof:</li><li>b) a second polynucleotide sequence encoding an integrase, a recombinase, or a transposase, or a portion thereof; and</li><li>c) a third polynucleotide sequence encoding a nucleic acid linker;</li></ol> wherein the first polynucleotide sequence comprises a 5' and a 3' end and the second polynucleotide sequence comprises a 5' and a 3' end, and the 3' end of the first polynucleotide is connected to the 5' end of the second polynucleotide by the nucleic acid linker, and the first and second polynucleotide are able to be expressed as a fusion protein in a cell or an organism.</li><li>9. A fusion protein, comprising: <ol id="ol0007" compact="compact"><li>a) a first protein that is a catalytically inactive Cas9, Cas9, a TALE protein, a Zinc finger protein, or a Cpfl protein, wherein the first protein is targeted to a target DNA sequence;</li><li>b) a second protein that is an integrase, a recombinase, or a transposase; and</li><li>c) a linker linking the first protein to the second protein.</li></ol></li><li>10. The fusion protein of aspect 9, wherein the second protein is an integrase.</li><li>11. The fusion protein of aspect 10, wherein the integrase is an HIV1 integrase or a lentiviral integrase.</li><li>12. The fusion protein of aspect 9, wherein the linker sequence is one or more amino acids in length.</li><li>13. The fusion protein of aspect 9, wherein the first protein is a catalytically inactive Cas9.</li><li>14. The fusion protein of aspect 9, wherein the linker sequence is 4-8 amino acids in length.</li><li>15. The fusion protein of aspect 14, wherein the first protein is a TALE protein.</li><li>16. The fusion protein of aspect 9, wherein the first protein is a Zinc finger protein.</li><li>17. The fusion protein of aspects 15 or 16, wherein the target DNA sequence is about 16 to about 24 base pairs in length.</li><li>18. The fusion protein of aspect 9, wherein the first protein is Cas9 or a catalytically inactive Cas9, and wherein one or more guide RNAs are used for targeting of a target DNA sequence of from about 16 to about 24 base pairs.</li><li>19. A method of inserting a DNA sequence into genomic DNA, comprising: <ul id="ul0021" list-style="none" compact="compact"><li>a) identifying a target sequence in the genomic DNA;</li><li>b) designing a fusion protein according to aspect 1 to bind to the target sequence in the genomic DNA;</li><li>3) designing a DNA sequence of interest to incorporate into the genomic DNA; and</li><li>d) providing the fusion protein and the DNA sequence of interest to a cell or organism by techniques that allow for entry of the fusion protein and DNA sequence of interest into the cell or organism; wherein the DNA sequence of interest becomes integrated at the target sequence in the genomic DNA.</li></ul></li><li>20. A nucleotide vector, comprising: <ol id="ol0008" compact="compact"><li>a) a first coding sequence for a first protein that is a Cas9, a catalytically inactive Cas9, a TALE protein, a Zinc finger protein, or a Cpf1 protein engineered to bind a target DNA sequence;</li><li>b) a second coding sequence for a second protein that is an integrase, a recombinase, or a transposase;</li><li>c) a DNA sequence between the first and second coding sequences that forms an amino acid linker between the first and second proteins;</li><li>d) optionally an expressed DNA sequence of interest surrounded by att sites recognized by an integrase, and optionally one or more guide RNAs, wherein the first protein is targeted to a determined DNA sequence, and wherein the first protein is linked to the second protein by the amino acid linker sequence.</li></ol></li><li>21. A method of inhibiting gene transcription in a cell or organism, comprising: <ol id="ol0009" compact="compact"><li>a) identifying an ATG start codon in a gene;</li><li>b) designing a fusion protein system with a fusion protein according to aspect 1 to bind to a target sequence immediately after the ATG start codon of the gene;</li><li>c) designing a DNA sequence of interest that is one or more consecutive stop codons; and</li><li>d) providing the fusion protein and the DNA sequence of interest to a cell or organism by techniques that allow for entry of the fusion protein and DNA sequence of interest into the cell or organism; wherein the DNA sequence of interest becomes integrated at the target sequence in the genomic DNA; and wherein transcription of the gene is inhibited.</li></ol></li><li>22. The fusion protein of aspect 9, wherein the second protein is a recombinase.</li><li>23. The fusion protein of aspect 22, wherein the recombinase is a Cre recombinase or a modified version thereof, wherein the modified Cre recombinase has constitutive recombinase activity.</li><li>24. The vector of aspect 20, further comprising a reverse transcriptase gene to be expressed in a cell.</li><li>25. A composition, comprising a purified protein of a DNA binding protein/integrase fusion and an RNA from about 15 to about 100 base pairs in length, wherein the DNA binding protein is selected from Cas9, Cpfl, a TALEN and a Zinc finger protein engineered to a targeted DNA sequence in a genome, and wherein the integrase is a HIV integrase, lentiviral integrase, adenoviral integrase, a retroviral integrase, or a MMTV integrase.</li></ol>