WO2017165859A1 - Modified viral capsid proteins - Google Patents

Abstract

Modified capsid proteins, isolated polynucleotides, methods for the preparation of modified capsid proteins, recombinant viral particles, recombinant expression systems for the generation of modified viral particles, and methods of gene editing are provided herein.

Description

MODIFIED VIRAL CAPSID PROTEINS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/313,006, filed March 24, 2016, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] The development of efficient and reliable ways to make specific, safe, and targeted changes to the genome of living cells with minimal off-target effects is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) has generated considerable excitement for its potential to efficiently perform gene editing.

[0003] Current CRISPR/Cas9 gene correction protocols suffer from a number of draw backs. Cas9 protein is a large enzyme that must be delivered efficiently to target tissues and cells to mediate gene repair through the CRISPR system. An additional guide RNA must usually be delivered via a separate vector due to packaging constraints. In addition, the optimal embodiment of the CRISPR/Cas9 system is to have the Cas9 protein only transiently expressed to reduce the chances of "off-target" gene rearrangements.

[0004] Another constraint with the CRISPR/Cas9 system is that one increases the risk for genetic modifications into alternative regions of the genome other than at the target site, due to Cas9 nicking sequences other than the gRNA target. These "off-target" sites may be critical to normal cell function and disruption of some regions may lead aberrant cell growth.

[0005] The present disclosure addresses the limitations of the prior art and provides related advantages as well.

SUMMARY

[0006] This disclosure relates to modified capsid proteins, isolated polynucleotides, methods for the preparation of modified capsid proteins, recombinant viral particles, recombinant expression systems for the generation of modified viral particles, and methods of gene editing. One aspect of the disclosure relates to a modified viral capsid protein that comprises, or alternatively consists essentially of, or yet further consists of, a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface of the viral capsid protein. [0007] Also disclosed herein is an isolated polynucleotide encoding a modified capsid protein that comprises, or alternatively consists essentially of, or yet further consists of, a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface of the viral capsid protein.

[0008] Provided herein is a method of preparing a modified capsid protein that comprises, or alternatively consists essentially of, or yet further consists of, a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface of the viral capsid protein. In one aspect, the method comprises coupling the Cas9 protein or an equivalent thereof to the viral capsid protein. Alternatively the method comprises expressing a recombinant fusion polynucleotide encoding Cas9 or an equivalent thereof and one or more viral capsid proteins, in a system that provides the helper functions for the preparation of viral particles. In one aspect, the viral particles are isolated from the system.

[0009] Also provided herein is a recombinant viral particle that comprises or alternatively consists essentially of, or yet further consists of, a modified capsid protein that comprises, or alternatively consists essentially of, or yet further consists of, a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface of the viral capsid protein, and one or more polynucleotides encapsulated within the capsid.

[0010] Further disclosed herein is a recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the viral particle surface, the system comprising, or alternatively consisting essentially of, or yet further consisting of: (a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or the equivalent thereof and a viral capsid protein; and (b) a helper plasmid.

[0011] Also disclosed herein is a method of gene editing comprising contacting a cell or tissue with a recombinant viral particle, the viral particle comprising, or alternatively consisting essentially of, or yet further consisting of, a modified capsid protein that comprises, or alternatively consists essentially of, or yet further consists of, a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface of the viral capsid protein, and one or more polynucleotides encapsulated within the viral capsid. The contacting can be in vitro (ex vivo) or in vivo.

[0012] This disclosure also provides compositions comprising a carrier and one or more of a modified protein, a polynucleotide, vector, plasmid, host cell, or expression system. Further provided is a kit comprising one or more of a modified protein, a polynucleotide, vector, plasmid, host cell, or expression system and instructions for use.

[0013] Further disclosed herein is a method of gene editing in a subject in need thereof, comprising, or alternatively consisting essentially of, or yet further consisting of,

administering to the subject an effective amount of a recombinant viral particle that comprises, or alternatively consists essentially of, or yet further consists of, a modified capsid protein that comprises, or alternatively consists essentially of, or yet further consists of, a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface of the viral capsid protein, and one or more polynucleotides encapsulated within the capsid.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 depicts two exemplary constructs: the first encoding VPsl and 3 of an AAV and the second encoding a VP2-Cas9 fusion protein.

[0015] FIG. 2 depicts four exemplary constructs: the first encoding VPsl and 3 of an AAV, the second encoding a VP2-Cas9 fusion protein, the third encoding a helper plasmid comprising the genes necessary for packaging the virus, and the fourth encoding a reporter gene (lucif erase) for detecting the virus.

[0016] FIG. 3 depicts a SYPRO stained gel from a crude cell lysate of a Cas9-VP2 virus preparation. The goal of this gel was to determine whether the large 193 kDa Cas9-VP2 fusion protein would be visible. This gel shows the abundance of VP1 and VP3 proteins in the gel.

[0017] FIG. 4 depicts a Western blot from HEK293 cells transfected with various plasmids. The plasmid in the first lane (after the ladder lane) is an AAV control2 plasmid that expresses normal AAV proteins (VP1, VP2, and VP3 which are approximately 87, 72, and 62 kDa respectively). The plasmid in lane 2 is a Cas9 control plasmid that expresses a Cas9 control protein of approximately 127 kDa. The plasmid in lane 3 is a VP 1-3 control2 plasmid that expresses only VP1 and VP3 proteins. The plasmid in lane 4 is a VP2-control2 plasmid that expresses only normal VP2 protein. The plasmid in lane 5 is VP2-Cas9 plasmid that expresses only Cas9-VP2 fusion proteins of approximately 193 kDa in size. The plasmid in lane 6 is VP2-cas9 help plasmid that expresses only Cas9-VP2 fusion protein and adenovirus helper proteins. The plasmid in lane 7 is Cas9 virus that expresses Cas9-VP2 fusion protein as well as VP1 and VP3 proteins. Cell ly sates were harvested 72 hours after transfection in RIPA buffer with protease inhibitors. Samples of each lysate were run on 4-12% gradient gel and probed with an anti-OLLAS antibody for the detection of OLLAS tagged Cas9 protein. Lane 2 shows a protein loading artifact with the sample which masked the detection of the positive control Cas9 protein. Lanes 5-7 clearly show the expression of the large Cas9-VP2 fusion protein as expected.

[0018] FIG. 5 depicts a Western blot of crude virus preparations of rh74-AVB control and Cas9 virus. Samples of each lysate were run on 4-12% gradient gel and probed with an anti- OLLAS antibody for the detection of OLLAS tagged Cas9 protein. Lane 2 shows a lower molecular weight protein than expected. This lower molecular weight band may be the result of protease degradation of the Cas9-VP2 fusion protein during the purification or may be non-specific binding of the anti-OLLAS antibody with the abundant VP3 protein which is also seen Western blots from crude lysate samples.

[0019] FIG. 6 depicts a Western blot from HEK293 cells transfected with the various plasmids listed below. In the first lane after the ladder, the plasmid is an AAV control2 plasmid that expresses normal AAV proteins (VP1, VP2, and VP3 which are approximately 87, 72, and 62 kDa respectively). The plasmid in lane 2 is a Cas9 control plasmid that expresses a Cas9 control protein of approximately 127 kDa. The plasmid in lane 3 is a VP 1-3 control2 plasmid that expresses only VP1 and VP3 proteins. The plasmid in lane 4 is a VP2- control2 plasmid that expresses only normal VP2 protein. The plasmid in lane 5 is VP2-Cas9 plasmid that expresses only Cas9-VP2 fusion proteins of approximately 193 kDa in size. The plasmid in lane 6 is VP2-cas9 help plasmid that expresses only Cas9-VP2 fusion protein and adenovirus helper proteins. The plasmid in lane 7 is Cas9 virus that expresses Cas9-VP2 fusion protein as well as VP1 and VP3 proteins. Cell ly sates were harvested 72 hours after transfection in RIPA buffer with protease inhibitors. Samples of each lysate were run on 4- 12% gradient gel and probed with an anti-AAV antibody for the detection of AAV proteins. Lane 2 shows a protein loading artifact with the sample. Lane 3 shows the expression of the most abundant VP3 protein as expected. The viral proteins in the positive control sample (lanel) and the viral proteins in lanes 4-7 were not abundant enough to detect in this image.

[0020] FIG. 7 depicts a Western blot of crude virus preps of rh74-AVB control and Cas9 virus probed with the anti-AAV antibody (Bl). Samples of each lysate were run on 4-12% gradient gel and probed with an anti-AAV antibody for the detection of AAV proteins. Lane 1 shows the correct size viral proteins from a purified prep of control AAVrh74 virus. Lane 2 shows a lower molecular weight protein. This lower molecular weight band is likely the most abundant VP3 protein that is affected by residual salts or proteins in the crude virus preparation that affected the migration.

[0021] FIG. 8 shows aliquots of chromatography fractions after purification before pooling and concentration. The samples are run on acrylamide gel and visualized with SYPRO stain. The virus fractions are too dilute to visualize the Cas9-VP2 fusion protein (193 kDa) and only the VP1 (87 kDa) and VP3 (62 kDa) proteins are visible.

[0022] FIG. 9 depicts an exemplary construct encoding a guide RNA under the control of a U6 promoter. The construct is pAV-U6-sgRNA-uDys.

[0023] FIG. 10 depicts a Western blot of crude virus preps. VP025 is a larger prep of virus that was purified via standard protocol. SAL Cas9 was a smaller virus prep where the cells were lysed after 72 hours and then purified by standard protocol to purify virus from inside the cell before release into the media. The OLLAS tag is only detecting proteins that contain the specific OLLAS tag sequence which would indicate the presence of lower molecular weight protein likely formed by protease cleavage during production or purification. A small amount of full length Cas9-VP2 protein is faintly visible. The Cas9 fusion protein is 193 kDa and the Cas9 alone is 127 kDa.

[0024] FIG. 11 depicts a Western blot of crude virus preps. VP025 is a larger prep of virus that was purified via standard protocol. SAL Cas9 was a smaller virus prep where the cells were lysed after 72 hours and then purified by standard protocol to purify virus from inside the cell before release into the media. The Bl antibody detects AAV specific capsid proteins. The AAVrh74 control virus lane shows the presence of all three virus capsid proteins whereas the VP025 and SAL Cas9 lanes only show the presence of VP3 and VP1 alone with some degradation proteins of lower molecular weight between 60-80 kDa.

DETAILED DESCRIPTION

[0025] Embodiments according to the present disclosure will be described more fully hereinafter. Aspects of the disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [0026] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. While not explicitly defined below, such terms should be interpreted according to their common meaning.

[0027] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0028] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0029] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0030] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.

[0031] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1 , as appropriate, or alternatively by a variation of +/- 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. [0032] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation or by an Arabic numeral. The full citation for the publications identified by an Arabic numeral are found immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this invention pertains.

Definitions

[0033] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g. , Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2^nd edition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0034] As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0035] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. As used herein, the transitional phrase consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the recited embodiment. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising." "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the present disclosure.

[0036] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount. [0037] The terms or "acceptable," "effective," or "sufficient" when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

[0038] Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of

combinations when interpreted in the alternative ("or").

[0039] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with this name and belonging to the genus dependoparvovirus, family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered, AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ. The AAV particle comprises three major viral proteins: VP1, VP2 and VP3.

[0040] The term "Cas9" refers to a CRISPR associated endonuclease referred to by this name. Non-limiting exemplary Cas9s are provided herein, e.g. the Cas9 provided for in UniProtKB G3ECR1 (CAS9_STRTR) or the Staphylococcus aureus Cas9 encoded by the protein sequence described herein, e.g., SEQ ID NO: 3, as well as the nuclease dead Cas9 encoded by the protein sequence SEQ ID NO: 40, orthologs and biological equivalents each thereof. Orthologs include but are not limited to Streptococcus pyogenes Cas9 ("spCas9"), e.g., SEQ ID NO: 18; Cas 9 from Streptococcus thermophiles , Legionella pneumophilia, Neisseria lactamica, Neisseria meningitides, Francisella novicida; and Cpfl (SEQ ID NO: 19) (which performs cutting functions analogous to Cas9) from various bacterial species including Acidaminococcus spp. and Francisella novicida U112.

[0041] The term "cell" as used herein may refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

[0042] "Eukaryotic cells" comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term "host" includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human, e.g., HEK293 cells and 293T cells.

[0043] "Prokaryotic cells" that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to

chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1 -2 μηι in diameter and 10 μηι long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0044] As used herein, the term "CRISPR" refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway, which unlike RNA interference regulates gene expression at a transcriptional level. The term "gRNA" or "guide RNA" as used herein refers to the guide RNA sequences used to target specific genes for correction employing the CRISPR technique. Techniques of designing gRNAs and donor therapeutic polynucleotides for target specificity are well known in the art. For example, Doench, J., et al. Nature biotechnology 2014; 32(12): 1262-7 and Graham, D., et al. Genome Biol. 2015; 16: 260, incorporated by reference herein.

[0045] The term "encode" as it is applied to nucleic acid sequences refers to a

polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0046] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological, or cellular material and intend those having minimal homology while still maintaining desired structure or functionality. Non-limiting examples of equivalent polypeptides, include a polypeptide having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity thereto or for polypeptide sequences, or a polypeptide which is encoded by a polynucleotide or its complement that hybridizes under conditions of high stringency to a polynucleotide encoding such polypeptide sequences. Conditions of high stringency are described herein and incorporated herein by reference. Alternatively, an equivalent thereof is a polypeptide encoded by a polynucleotide or a complement thereto, having at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity, or at least 97% sequence identity to the reference polynucleotide, e.g., the wild-type polynucleotide.

[0047] Non-limiting examples of equivalent polypeptides, include a polynucleotide having at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 97%, identity to a reference polynucleotide. An equivalent also intends a polynucleotide or its complement that hybridizes under conditions of high stringency to a reference polynucleotide.

[0048] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al, eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;

Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS

translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST. Sequence identity and percent identity can determined by incorporating them into clustalW (available at the web address: genome.jp/tools/clustalw/, last accessed on Jan. 13, 2017).

[0049] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0050] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0051] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0052] Examples of stringent hybridization conditions include: incubation temperatures of about 25° C. to about 37° C; hybridization buffer concentrations of about 6*SSC to about lOxSSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4*SSC to about 8xSSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40° C. to about 50° C; buffer concentrations of about 9*SSC to about 2*SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5*SSC to about 2*SSC. Examples of high stringency conditions include: incubation temperatures of about 55° C. to about 68° C; buffer concentrations of about 1 xSSC to about 0.1 xSSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 xSSC, 0.1 xSSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[0053] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

[0054] The term "isolated" as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. [0055] As used herein, the term "functional" may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.

[0056] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either

ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0057] The term "promoter" as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters may be constitutive, inducible, repressible, or tissue-specific, for example. A "promoter" is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include CMV promoter (e.g., SEQ ID NO: 41, base pairs numbered 140 to 774 of SEQ ID NO: 7, or an equivalent of each thereof), and U6 promoter (e.g., SEQ ID NO: 42, base pairs numbered 4404 to 4395 of SEQ ID NO: 8, or an equivalent of each thereof). Additional non-limiting exemplary promoters with certain target specificity are provided herein below including but not limited to CMV, EFla, SV40 (e.g., base pairs numbered 3434-3702 of SEQ ID NO: 7), PGKl (human or mouse), P5 (e.g., base pairs numbered 10749 to 10828 of SEQ ID NO: 5), Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, Gall, 10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, and Alpha- 1 -antitrypsin. Synthetically-derived promoters may be used for ubiquitous or tissue specific expression. Further, virus-derived promoters, some of which are noted above, may be useful in the methods disclosed herein, e.g., CMV, HIV, adenovirus, and AAV promoters.

[0058] The term "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. [0059] As used herein, the term "recombinant expression system" refers to a genetic construct or constructs for the expression of certain genetic material formed by

recombination.

[0060] A "gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0061] A polynucleotide disclosed herein can be delivered to a cell or tissue using a gene delivery vehicle. "Gene delivery," "gene transfer," "transducing," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector- mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.

[0062] A "plasmid" is an extra-chromosomal DNA molecule separate from the

chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. [0063] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene.

[0064] A "yeast artificial chromosome" or "YAC" refers to a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearized by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends. Yeast expression vectors, such as YACs, Yips (yeast integrating plasmid), and YEps (yeast episomal plasmid), are extremely useful as one can get eukaryotic protein products with posttranslational modifications as yeasts are themselves eukaryotic cells, however YACs have been found to be more unstable than BACs, producing chimeric effects.

[0065] A "viral vector" is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.

[0066] Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors, alphavirus vectors and the like. Infectious tobacco mosaic virus (TMV)-based vectors can be used to manufacturer proteins and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106(15):6099- 6104). Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus- based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7): 823-827. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. Further details as to modern methods of vectors for use in gene transfer may be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. [0067] As used herein, "retroviral mediated gene transfer" or "retroviral transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

[0068] Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0069] In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses (Ads) are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. Ads do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. Such vectors are commercially available from sources such as Takara Bio USA (Mountain View, CA), Vector Biolabs (Philadelphia, PA), and Creative Biogene (Shirley, NY). Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Wold and Toth (2013) Curr. Gene. Ther.

13(6):421-433, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470, and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0070] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression. [0071] Gene delivery vehicles also include DNA/liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and/or promote the activity of the proteins disclosed herein are other non-limiting techniques.

[0072] As used herein the term "helper" in reference to a virus or plasmid refers to a virus or plasmid used to provide the additional components necessary for replication of a recombinant virus, such as the modified AAV disclosed herein. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus).

[0073] As used herein, the term "exterior" in reference to a viral capsid protein refers to the surface, domain, region, or terminal end of the capsid protein that is exterior-facing in an assembled viral capsid.

[0074] As used herein, the term "conjugated" refers to any method of attaching, coupling, or linking a viral capsid protein to a Cas9 protein or an equivalent thereof. Non-limiting examples of conjugation include recombinant fusion proteins wherein the Cas9 protein or an equivalent thereof and the viral capsid protein are encoded by a single polynucleotide that comprises the genes for both the Cas9 protein or an equivalent thereof and the viral capsid protein, posttranslational modification that causes a chemical bond to form between a Cas9 protein or equivalent thereof and the viral capsid protein, linkage of a Cas9 or equivalent thereof and a viral capsid protein via a linker, and coating an assembled viral capsid with Cas9 or an equivalent thereof.

[0075] As used herein, the term "label" intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluoresecence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0076] Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

[0077] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^th ed.).

[0078] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0079] Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to,

antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavidin.

[0080] The phrase "solid support" refers to non-aqueous surfaces such as "culture plates" "gene chips" or "microarrays." Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are attached and arrayed on a gene chip for determining the DNA sequence by the hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The polynucleotides of this invention can be modified to probes, which in turn can be used for detection of a genetic sequence. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659. A probe also can be attached or affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayem et al. U.S. Patent No. 5,952,172 and by Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

[0081] A "composition" is intended to mean a combination of active polypeptide, polynucleotide or antibody and another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0082] A "pharmaceutical composition" is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0083] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton ).

[0084] A "subject" of diagnosis or treatment is a cell or an animal such as a mammal, or a human. A subject is not limited to a specific species and includes non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals, and pets. Human patients are included within the term as well. [0085] The term "tissue" is used herein to refer to tissue of a living or deceased organism or any tissue derived from or designed to mimic a living or deceased organism. The tissue may be healthy, diseased, and/or have genetic mutations. The biological tissue may include any single tissue (e.g., a collection of cells that may be interconnected) or a group of tissues making up an organ or part or region of the body of an organism. The tissue may comprise a homogeneous cellular material or it may be a composite structure such as that found in regions of the body including the thorax which for instance can include lung tissue, skeletal tissue, and/or muscle tissue. Exemplary tissues include, but are not limited to those derived from liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidneys, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart and intestines, including any combination thereof.

[0086] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its

development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable.

[0087] A number of effector elements are disclosed herein. The nature and function of these effector elements are commonly understood in the art and a number of these effector elements are commercially available. Where relevant, non-limiting exemplary sequences thereof are disclosed herein and further description thereof is provided herein below.

Modes of Carrying Out the Disclosure

Modified Viral Capsids and Methods of Preparation

[0088] Disclosed herein is a modified viral capsid protein comprising, or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein. In some aspects, conjugation comprises or alternatively consists essentially of, or yet further consists of, a fusion protein, e.g. a fusion of a Cas9 protein or an equivalent thereof with a viral capsid protein wherein the Cas9 protein or an equivalent thereof is fused to the exterior surface of the viral capsid protein. In one aspect, the Cas9 or equivalent thereof is fused to the amino terminal end of the viral capsid protein. Non-limiting examples of fusions of Cas9 and VP2 include SEQ ID NO: 36, nucleotide base pairs numbered 5037 to 10565 of SEQ ID NO: 2, base pairs numbered 5532 to 10574 of SEQ ID NO: 5, and equivalents of each thereof. In other aspects, conjugation comprises or alternatively consists essentially of, or yet further consists of, posttranslational modifications that cause a bond between a viral capsid protein and a Cas9 or an equivalent thereof, e.g. covalent bonds, hydrogen bonds, or ionic bonds. In some aspects, conjugation comprises or alternatively consists essentially of, or yet further consists of, coating the exterior surface of assembled viral particles with a Cas9 or an equivalent thereof.

[0089] In a further aspect, the modified viral capsid as described herein, is coupled to a detectable label for ease of detection. Non-limiting examples of such labels are known in the art and described herein.

[0090] In one aspect, conjugation comprises or alternatively consists essentially of, or yet further consists of, attaching a Cas9 or equivalent thereof to the exterior surface of a viral capsid protein via a linker. In some aspects, the linkers are flexible or rigid. In one aspect, a biotin ligase is used to join the purified protein moiety with the purified viral preparation. Additional examples of conjugation of a protein with a capsid protein are described in Stachler et al. (2008) Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase. Mol. Ther. 16: 1467-1473,

doi: 10.1038/mt.2008.129, and Wei et al. (2012) Conjugation of paclitaxel on adeno- associated virus (AAV) nanoparticles for co-delivery of genes and drugs. Eur. J. Pharm. Sci. 46: 167-172, doi: 10.1016/j.ejps.2012.02.022, incorporated by reference herein.

[0091] In one aspect, a Cas9 protein or an equivalent thereof is conjugated to a viral capsid protein via a biotin linker. Escherichia coli enzyme biotin ligase (BirA), ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. Use of a ketone isotere of biotin as a cofactor allows for ligation of a peptide to a BAP-modified AAV capsid. Ketones are absent from AAV, allowing BAP-modified AAV particles to be tagged with the ketone peptide and then specifically conjugated to hydrazide- or hydroxylamine- functionalized molecules. [0092] In some aspects, the modified capsid protein further comprises, or alternatively consists essentially of, or yet further consists of, a spacer region between the Cas9 or an equivalent thereof and the viral capsid protein to minimize any steric hindrance on viral capsid assembly and/or formation. In one aspect, the spacer region comprises or altematively consists essentially of, or yet further consists of, a peptide. In some aspects, the peptide is between 1 and 100 amino acids in length, between 1 and 50 amino acids in length, between 1 and 30 amino acids in length, between 1 and 20 amino acids in length, between 1 and 10 amino acids in length, between 1 and 5 amino acids in length, between 5 and 10 amino acids in length, between 5 and 15 amino acids in length, or between 20 and 40 amino acids in length. In one aspect, the spacer region is encoded by a polynucleotide comprising SEQ ID NO: 9 or an equivalent thereof. As used herein, a "spacer" includes a peptide sequence comprising a linker. Nonlimiting examples of flexible linkers include

KESGSVSSEQLAQFRSLD (SEQ ID NO: 31) and EGKSSGSGSESKST (SEQ ID NO: 32), which have been applied for the construction of a bioactive scFv (Bird, R. E. et al. Science 242, 423-426 (1988), incorporated by reference herein). Additional examples of linkers include but are not limited to (Gly)8 (SEQ ID NO: 33), consisting of glycine residues, GS AGS AAGS GEF (SEQ ID NO: 34), an empirical rigid linker with the sequence of A(EAAAK)n A (n = 2-5) (SEQ ID NO: 35) and a linker with a -helical conformation and stabilized by the Glu- -Lys+ salt bridges within segments. Additional methods of producing linkers and descriptions of the above linkers are found, for example, in Sabourin, M. et al. (2007) Yeast 24:39-45, doi: 10.1002/yea. l431; Waldo, GS. et al. (1999) Nat Biotechnol. 17:691-695, doi: 10.1038/10904 (1999); Arai et al. (2001) Protein Eng. 14:529-532; and Arai et al. (2004) Proteins 57:829-838, incorporated by reference herein.

[0093] In one aspect, the viral capsid protein is selected from the group of an adenoviral (Ad) capsid protein, an adeno-associated virus (AAV) capsid protein, or a lentivirus capsid or envelope protein. Non-limiting examples of Ad capsid proteins include hexon (protein II), penton base (protein III) and fibre (protein IV) and proteins Ilia, VI, VIII and IX or an equivalent of each thereof. These sequences are known in the art and described for example in Athappilly FK, et al., J Mol Biol 1994;242:430-455. Non-limiting examples of AAV viral proteins include VP1 (SEQ ID NO: 37), VP2 (SEQ ID NO: 39), and VP3 (SEQ ID NO: 38), or an equivalent of each thereof. Nonlimiting examples of lentiviral capsid and envelope proteins include P24 capsid protein CA and P9 capsid protein NC, VSVG and equivalents of each thereof. In one aspect, the modified viral capsid protein comprises AAV VP2, or an equivalent thereof.

[0094] In some aspects, the Cas9 protein is a S. aureus Cas9 (SEQ ID NO: 3) or an equivalent thereof. In other aspects, the Cas9 protein is a Streptococcus pyogenes (SP) SpCas9 with the PAM sequence NGG (SEQ ID NO: 18), SpCas9 Dl 135E variant with the PAM sequence NGG (reduced NAG binding), SpCas9 VRER variant with the PAM sequence NGCG, SpCas9 EQR variant with the PAM sequence NGAG, SpCas9 VQR variant with PAM sequences NGAN or NGNG, Staphylococcus aureus (SA) SaCas9 with PAM sequences NNGRRT or NNGRR(N), Neisseria meningitidis (NM) Cas9 with the PAM sequence of NNNNGATT, Streptococcus thermophilus (ST) Cas9 with the PAM sequence NNAGAAW, Treponema denticola (TD) Cas9 with the PAM sequence NAAAAC, or a Cas protein from another bacterial species such as Prevotella, Acidaminococcus,

Lachnospiraceae, or Francisella. Equivalents of Cas9 include but are not limited to Cas9s with modifications that affect the protein's function, targeting specificity, size, localization, and/or reduce off-target effects such as a nuclease dead Cas9 (dCas9, SEQ ID NO: 40) that is enzymatically inactive but can bind but cannot cleave DNA, a Cas9 nickase (Cas9n) in which one of the two nuclease domains are inactivated (either RuvC or HNH) rendering the enzyme capable of cleaving only one strand of target DNA, a nuclease dead Cas9 fused to the nonspecific endonuclease Fokl (dCas9-Fokl), spCas9 VQR, EQR and VRER variants that recognize novel NGG PAM sequences, and non-Cas9 CRISPR endonuclease Cpfl which leaves a 5 nucleotide 5' overhang 18 base pairs from the PAM sequence when cleaving DNA (SEQ ID NO: 10). In some aspects, the Cas9 protein comprises or consists of SEQ ID NO: 3, or an equivalent thereof. In some aspects, Cas9 can be modified to be resistant to protease degradation or cleavage. Methods for designing protease resistant proteins are known in the art, as described in Fruchart-Gaillard, C. et al. (2012) PLoS One 7:e39166; Hu, W. et al. Enzyme Microb Technol 97, 82-89 (2017); Kukenshoner, T. et al. (2014) J Struct Biol 186:335-348 (2014); Li, Y. et al. (2013) J Biotechnol. 163:401-407; and Werner, H.M. et al. (2016) Chembiochem 17:712-718, incorporated by reference herein.

[0095] In some aspects, the disclosure provides one or more isolated polynucleotides encoding a modified viral capsid protein comprising, or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein. In one aspect, the polynucleotide encodes a fusion protein wherein a single polynucleotide comprises or alternatively consists essentially of, or yet further consists of a polynucleotide encoding a Cas9 protein or an equivalent thereof and a polynucleotide encoding a viral capsid protein. In a further aspect, the polynucleotide encoding a fusion protein further comprises a polynucleotide sequence encoding a spacer region and/or linker between the Cas9 or an equivalent thereof and the viral capsid protein. In one aspect, the Cas9 encoded by the polynucleotide is saCas9 and the viral capsid protein encoded by the polynucleotide is VP2. In another aspect, the polynucleotide encodes the Cas9 protein comprising or consisting of SEQ ID NO: 3. In other aspects, two or more distinct polynucleotides encode the Cas9 protein or an equivalent thereof and the capsid protein. In some aspects, the polynucleotide encoding the Cas9 and/or viral capsid protein is codon-optimized for expression in humans.

[0096] In a further aspect, the polynucleotides are operatively coupled to regulatory sequences necessary for the replication and/or expression, e.g., a promoters and optionally enhancers. Non-limiting examples of such are disclosed herein, e.g., U6 promoter.

[0097] In a further aspect, the polynucleotides are contained within a gene expression vehicle, a vector, such as a viral vector or plasmid. Non-limiting examples are known in the art and briefly described herein. As is apparent to the skilled artisan, the polynucleotides are contained in the gene expression vehicles in the appropriate orientation for expression of the polynucleotides.

[0098] In a further aspect, the polynucleotides are attached to a detectable label. Non- limiting examples of labels are described herein.

[0099] In a further aspect, the two or more distinct polynucleotides are on the same or different plasmids. In one aspect, the isolated polynucleotide comprised or consists of SEQ ID NO: 5. In yet another aspect, one of the two distinct polynucleotides further comprises a spacer region and/or linker.

[0100] In addition, provided herein is a vector or host cell comprising the one or more isolated polynucleotides encoding a modified viral capsid protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein. In some aspects, the vector or host cell further comprises additional plasmids necessary for the production and assembly of viral particles and/or plasmids encoding components for gene editing. Non-limiting examples of vectors or host cells include HEK293 cells, 293T cells, or an equivalent of each thereof, commercially available viral packaging cells, e.g., 293 AAV cells (Cell Biolabs, Inc.) or Phoenix packaging cells (ATTC). In some aspects, the vector or host cells further comprise a helper plasmid encoding genes necessary for viral packaging.

[0101] Some aspects of this disclosure relate to methods of preparing a modified viral capsid protein comprising, or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein, the method comprising or alternatively consisting essentially of, or yet further consisting of, coupling the Cas9 protein or an equivalent thereof to the viral capsid protein. In some aspects, coupling comprises or alternatively consists essentially of, or yet further consists of, posttranslational modifications that cause a bond between a viral capsid protein and a Cas9 or an equivalent thereof, e.g. covalent bonds, hydrogen bonds, or ionic bonds. In some aspects, coupling comprises or alternatively consists essentially of, or yet further consists of, coating the exterior surface of assembled viral particles with a Cas9 or an equivalent thereof. In one aspect, coupling comprises or alternatively consists essentially of, or yet further consists of, attaching a Cas9 or equivalent thereof to the exterior surface of a viral capsid protein via a linker. In some aspects, the linkers are flexible or rigid.

[0102] Some aspects of this disclosure provide a method of preparing a modified viral capsid protein, the protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein, the method comprising or alternatively consisting essentially of, or yet further consisting of, expressing one or more isolated polypeptide encoding the modified viral capsid protein. In one aspect, the isolated polypeptide comprises or consists of SEQ ID NO: 5.

Modified Viral Particles Expressing Cas9 on the Exterior Capsid Surface

[0103] Also provided herein are recombinant or modified viral particles comprising or alternatively consisting essentially of a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein and one or more polynucleotides encapsulated within the capsid. In some aspects, at least one of the polynucleotides comprises or consists essentially of, or yet further consists of a polynucleotide encoding a guide RNA (gRNA). In some aspects, at least one of the polynucleotides comprises or alternatively consists essentially of, or yet further consists of a therapeutic polynucleotide. As used herein, the term "therapeutic polynucleotide" intends a replacement polynucleotide that can be for genetic modification of a target cell genome. Alternatively the therapeutic polynucleotide encodes a therapeutic polypeptide.

[0104] In some aspects, the polynucleotide encoding the gRNA comprises or alternatively consists essentially of, or yet further consists of a fusion polypeptide comprising CRISPR RNA (crRNA) and trans -activating CRIPSPR RNA (tracrRNA); or a polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA). In one aspect, the polynucleotide encoding the gRNA comprises or consists of SEQ ID NO: 8 or an equivalent thereof. In some aspects, the gRNA is specific for a region of DNA that is in need of gene editing. In a further aspect the gRNA contains a detectable label.

[0105] In some aspects, the recombinant viral particle further comprising a therapeutic polynucleotide. The therapeutic polynucleotide is any polypeptide that can be used to target a DNA sequence in need of editing, provide a repair template for a DNA sequence in need of editing, or provide a replacement for a DNA sequence in need of editing. In further aspects, the therapeutic polypeptide comprises a wild-type sequence of a gene in need of editing. In a further aspect the therapeutic polynucleotide contains a detectable label.

[0106] Disclosed herein is a recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the viral particle surface, the system comprising or alternatively consisting essentially of, or yet further consisting of (a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or the equivalent thereof and a viral capsid protein; and (b) a helper plasmid. In some aspects, the viral capsid is selected from the group of an adenoviral (Ad) capsid protein, an adeno-associated virus (AAV) capsid protein, or a lentivirus. Non-limiting examples of Ad capsid proteins include hexon (protein II), penton base (protein III) and fibre (protein IV) and proteins Ilia, VI, VIII and IX or an equivalent of each thereof. Non-limiting examples of AAV viral proteins include VP1, VP2, and VP3, or an equivalent of each thereof. Non limiting examples of VP1 include SEQ ID NO: 37, DNA base pairs numbered 5037 to 7253 of SEQ ID NO: 1, base pairs numbered 5037 to 7253 of SEQ ID NO: 4, and equivalents of each thereof. Nonlimiting examples of VP2 include SEQ ID NO: 39, base pairs numbered 8786 to 10574 of SEQ ID NO: 5, and equivalents of each thereof.

Nonlimiting examples of VP3 include SEQ ID NO: 38, base pairs numbered 5646 to 7253 of SEQ ID NO: 1, base pairs numbered 5646 to 7253 of SEQ ID NO: 1, and an equivalent of each thereof. Non-limiting examples of lentiviral capsid proteins include P24 capsid protein CA, P9 capsid protein NC, lentiviral envelope protein VSVG, and equivalents of each thereof. In some aspects, the modified capsid protein comprises one or more of AAV VP1, VP2, and VP3, or an equivalent of each thereof. In one aspect, the modified viral capsid protein comprises VP2, or an equivalent thereof. Non-limiting examples of Ad capsid proteins include hexon (protein II), penton base (protein III) and fibre (protein IV) and proteins Ilia, VI, VIII and IX or an equivalent of each thereof. Non-limiting examples of AAV viral proteins include VP1, VP2, and VP3, or an equivalent of each thereof. Non- limiting examples of lentiviral capsid proteins include P24 capsid protein CA and P9 capsid protein NC and equivalents of each thereof.

[0107] In some aspects, the Cas9 protein is a S. aureus Cas9 or an equivalent thereof. In other aspects, the Cas9 protein is a Streptococcus pyogenes (SP) SpCas9 with the PAM sequence NGG, SpCas9 Dl 135E variant with the PAM sequence NGG (reduced NAG binding), SpCas9 VRER variant with the PAM sequence NGCG, SpCas9 EQR variant with the PAM sequence NGAG, SpCas9 VQR variant with PAM sequences NGAN or NGNG, Staphylococcus aureus (S A) SaCas9 with PAM sequences NNGRRT or NNGRR(N), Neisseria meningitidis (NM) Cas9 with the PAM sequence of NNNNGATT, Streptococcus thermophilus (ST) Cas9 with the PAM sequence NNAGAAW, Treponema denticola (TD) Cas9 with the PAM sequence NAAAAC, or a Cas protein from another bacterial species such as Prevotella, Acidaminococcus, Lachnospiraceae, or Francisella. In the above sequences, N stands for any nucleotide. Equivalents of Cas9 include but are not limited to Cas9s with modifications that affect the protein's function, targeting specificity, size, localization, and/or reduce off-target effects such as a nuclease dead Cas9 (dCas9) that is enzymatically inactive but can bind but cannot cleave DNA, a Cas9 nickase (Cas9n) in which one of the two nuclease domains are inactivated (either RuvC or HNH) rendering the enzyme capable of cleaving only one strand of target DNA, a nuclease dead Cas9 fused to the non-specific endonuclease Fokl (dCas9-Fokl), spCas9 VQR, EQR and VRER variants that recognize novel NGG PAM sequences, and non-Cas9 CRISPR endonuclease Cpfl which leaves a 5 nucleotide 5' overhang 18 base pairs from the PAM sequence when cleaving DNA. In some aspects, the Cas9 protein comprises or consists of SEQ ID NO: 3, or an equivalent thereof. [0108] In some aspects, recombinant expression system comprises a fusion protein comprising or alternatively consisting essentially of, or yet further consisting of, Cas9 and VP2. In additional aspects, the recombinant expression system comprises or alternatively consists essentially of, or yet further consists of a plasmid comprising or consisting of a DNA sequence selected from the group of SEQ ID NO: 2, SEQ ID NO: 5, or an equivalent of each thereof. In some aspects, the recombinant expression system comprises or alternatively consists essentially of, or yet further consists of a helper plasmid comprising or consisting of a DNA sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 4, or an equivalent of each thereof. In a further aspect, the helper plasmid comprises or consists of SEQ ID NO: 6 or an equivalent thereof. In some aspects, the recombinant expression system comprises or alternatively consists essentially of, or yet further consists of a DNA sequence selected from the group of a DNA sequence encoding VP2, a DNA sequence encoding Cas9, a DNA sequence encoding SEQ ID NO: 36, or an equivalent of each thereof. In some aspects, the recombinant expression system comprises a helper plasmid comprising a DNA sequence selected from the group of a DNA sequence encoding VP1, a DNA sequence encoding VP3, or a DNA sequence encoding both VP1 and VP3, or an equivalent of each thereof.

[0109] The modified virus, e.g., AAV can be packaged into a retroviral packaging system by using a helper virus or helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that facilitate the delivery of genetic materials into cells. In another aspect, the helper plasmid or a polynucleotide comprising the helper plasmid is stably incorporated into the genome of a packaging cell line, such that the packaging cell line does not require additional transfection with a helper plasmid.

[0110] A helper plasmid may comprise, for example, at least one retroviral helper DNA sequence derived from a replication-incompetent retroviral genome encoding in trans all virion proteins required to package a replication incompetent AAV, and for producing virion proteins capable of packaging the replication-incompetent AAV at high titer, without the production of replication-competent AAV. The retroviral DNA sequence lacks the region encoding the native enhancer and/or promoter of the viral 5' LTR of the virus, and lacks both the psi function sequence responsible for packaging helper genome and the 3' LTR, but encodes a foreign polyadenylation site, for example the SV40 polyadenylation site, and a foreign enhancer and/or promoter which directs efficient transcription in a cell type where virus production is desired. The retrovirus is a leukemia virus such as a Moloney Murine Leukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or the Gibbon Ape Leukemia virus (GALV). The foreign enhancer and promoter may be the human

cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the enhancer and promoter (U3 region) of the Moloney Murine Sarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3 region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancer joined to the native Moloney Murine Leukemia Virus (MMLV) promoter. The helper plasmid may consist of two retroviral helper DNA sequences encoded by plasmid based expression vectors, for example where a first helper sequence contains a cDNA encoding the gag and pol proteins of ecotropic MMLV or GALV and a second helper sequence contains a cDNA encoding the env protein. The Env gene, which determines the host range, may be derived from the genes encoding xenotropic, amphotropic, ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virus env proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, the Human Immunodeficiency Virus env (gpl60) protein, the Vesicular Stomatitus Virus (VSV) G protein, the Human T cell leukemia (HTLV) type I and II env gene products, chimeric envelope gene derived from combinations of one or more of the aforementioned env genes or chimeric envelope genes encoding the cytoplasmic and transmembrane of the aforementioned env gene products and a monoclonal antibody directed against a specific surface molecule on a desired target cell.

[0111] In the packaging process, the helper plasmids and the plasmids encoding the AAV viral proteins are transiently cotransfected into a first population of mammalian cells that are capable of producing virus, such as human embryonic kidney cells, for example 293 cells (ATCC No. CRL1573, ATCC, Rockville, Md.) to produce high titer recombinant retrovirus- containing supernatants. In another method of the invention this transiently transfected first population of cells is then cocultivated with mammalian target cells, for example human lymphocytes, to transduce the target cells with the foreign gene at high efficiencies.

[0112] In another aspect, the helper plasmids are stably expressed in a first population of mammalian cells that are capable of producing virus, such as human embryonic kidney cells, for example 293 cells. The plasmids are introduced into cells in an episomally maintained plasmid. High titer modified AAV containing supernatants are produced, and the modified AAV may be purified out or maintained in this high titer supernant for use in the methods of treatment disclosed herein below.

[0113] In further aspects, the recombinant expression system further comprises a polynucleotide encoding one or more guide RNAs. In other aspects, the recombinant expression system further comprises a therapeutic polynucleotide. [0114] Also disclosed herein is a method of producing modified AAV expressing Cas9 or an equivalent thereof on its surface comprising transfecting one or more cells with a recombinant expression system consisting essentially of, or yet further consisting of (a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or the equivalent thereof and a viral capsid protein; and (b) a helper plasmid. In some aspects, the method comprises an HEK293 or a similar cell transfected with plasmids that encode for VP1 + VP3 and, in a separate reading frame, the Cas9-VP2 fusion protein. In addition, the targeting vector containing the guide RNA sequence and, if needed, additional therapeutic polynucleotide. In another aspect, the method further comprises transfection of the HEK or similar cell with an additional helper plasmid that provides the viral helper function found in Adenovirus (E1A, E1B, E2A, E40RF6 and VA RNAs) or Herpes virus (among other viruses as well) to enable efficient AAV production. The AAV and Helper genes can be provided as separate plasmids or combined into multiples or a single plasmid if desired. The genes can be stably introduced into cells to generate stable packing cell lines in another embodiment. Alternatively, the genes can be introduced into cells using viral vectors like baculo-virus or herpes virus to amplify and deliver large quantities of the needed genes to adherent or suspension grown cells.

[0115] Provided herein is a modified AAV particle expressing Cas9 or an equivalent thereof on its surface produced by a method of transfecting one or more cells with a recombinant expression system consisting essentially of, or yet further consisting of (a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or the equivalent thereof and a viral capsid protein; and (b) a helper plasmid. In some aspects, the AAV particle comprises Cas9 or an equivalent thereof conjugated to the exterior of VP2. In other aspects, the AAV particle comprises Cas9 or an equivalent thereof conjugated to VP1 or VP3.

[0116] The present disclosure relates to a modified adeno-associated virus (AAV) expressing Cas9 on its surface and methods of making and using said modified AAV. A non- limiting examples of such are disclosed herein, as well as biological equivalents of such. Non-limiting example of a suitable biological equivalents include a polynucleotide having at least 70%, or alternatively 75 %, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95 % sequence identity of the various elements. [0117] Aspects of the disclosure relate to a modified adeno-associated virus (AAV) expressing Cas9 on its surface comprising an AAV viral protein selected from the group of VP1, VP2, and VP3 fused to Cas9. In some embodiments, the AAV viral protein is VP2. In some embodiments, the Cas9 is S. aureus Cas9. In further embodiments, the Cas9 comprises the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, the modified AAV comprises and/or encapsulates one or more guide RNAs or polynucleotides encoding said guide RNAs.

[0118] Further aspects of the disclosure relate to a recombinant expression system for the generation of such a modified AAV. In some embodiments the recombinant expression system comprises a plurality of plasmids; the plurality encoding all of the AAV viral proteins - VP1, VP2, and VP3. In some embodiments, each viral protein is encoded in a different plasmid. In some embodiments, one or more viral proteins is encoded in the same plasmid. In some embodiments, at least one viral protein is encoded as a fusion protein with Cas9.

[0119] Accordingly, embodiments disclosed herein relate to a recombinant expression system for the generation of a modified AAV expressing Cas9 on its surface comprising: (a) a plasmid comprising a DNA sequence encoding a fusion protein comprising Cas9 and an AAV viral protein selected from the group of VP1, VP2, and VP3, and (b) a plasmid comprising a DNA sequence encoding any AAV viral proteins selected from the group of VP1, VP2, and VP3 not comprised in the fusion protein of plasmid (a). In some

embodiments, the fusion protein comprises VP2. In some embodiments, the Cas9 is S. aureus Cas9. In further embodiments, the Cas9 comprises the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, plasmid (a) comprises a DNA sequence selected from the group of SEQ ID NO: 2 and SEQ ID NO: 5. In some embodiments, plasmid (b) comprises a DNA sequence selected from the group of SEQ ID NO: 1 and SEQ ID NO: 4. In some embodiments, the recombinant expression system further comprises a helper virus or helper plasmid. In some embodiments, the helper plasmid comprises the DNA sequence provided in SEQ ID NO: 6. In some embodiments, the recombinant expression further comprises a plasmid comprising a DNA sequence encoding one or more guide RNAs.

[0120] Some aspects of the disclosure relate to methods of producing the modified AAVs using the recombinant expression system disclosed herein. Aspects relate to a method of producing a modified AAV expressing Cas9 on its surface by transfecting one or more cells with the recombinant expression system disclosed herein. In some embodiments, the one or more cells are HEK293 cells. Compositions

[0121] Also provided by this invention is a composition or kit comprising any one or more of the viral vectors, isolated cells, packaging system, viral particles as described herein and a carrier. In one aspect, the carrier is a pharmaceutically acceptable carrier. These compositions can be used therapeutically as described herein and can be used in combination with other known therapies.

Methods of Administering Modified Viral Particles

[0122] Provided herein is a non-human transgenic animal comprising a modified viral capsid protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein. Also provided herein is a non-human transgenic animal comprising a modified or recombinant viral particle comprising or alternatively consisting essentially of a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein and one or more polynucleotides encapsulated within the capsid.

[0123] Disclosed herein is a method of gene editing comprising contacting a cell with recombinant viral particle comprising or alternatively consisting essentially of a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior-facing terminal end of the viral capsid protein and one or more polynucleotides encapsulated within the capsid. In some aspects, the contact is in vitro. In other aspects, the contact is in vivo. In some aspects, the contact is in vivo or in vitro. In some aspects, at least one of the polynucleotides comprises or consists essentially of, or yet further consists of a polynucleotide encoding a guide RNA (gRNA). In some aspects, at least one of the polynucleotides comprises or alternatively consists essentially of, or yet further consists of a therapeutic polypeptide.

[0124] Further disclosed herein is a method of gene editing in a subject in need thereof, comprising administering to the subject an effective amount recombinant viral particle comprising or alternatively consisting essentially of a modified capsid wherein the modified capsid comprises a modified viral capsid protein comprising or alternatively consisting essentially of, or yet further consisting of a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior surface, exterior facing domain, or the exterior- facing terminal end of the viral capsid protein and one or more polynucleotides encapsulated within the capsid. In some aspects, at least one of the polynucleotides comprises or consists essentially of, or yet further consists of a polynucleotide encoding a guide RNA (gRNA). In some aspects, at least one of the polynucleotides comprises or alternatively consists essentially of, or yet further consists of a therapeutic polypeptide.

[0125] In some aspects, the polynucleotide encoding the gRNA comprises or alternatively consists essentially of, or yet further consists of a fusion polypeptide comprising CRISPR RNA (crRNA) and trans -activating CRIPSPR RNA (tracrRNA); or a polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA). In one aspect, the polynucleotide encoding the gRNA comprises or consists of SEQ ID NO: 8 or an equivalent thereof. In some aspects, the gRNA is specific for a region of DNA that is in need of gene editing in the subject or cell in need thereof.

[0126] In some aspects, the recombinant viral particle further comprising a therapeutic polynucleotide. The therapeutic polynucleotide is any polypeptide that can be used to target a DNA sequence in need of editing, provide a repair template for a DNA sequence in need of editing, or provide a replacement for a DNA sequence in need of editing. In further aspects, the therapeutic polypeptide comprises a wild-type sequence of a gene in need of editing in the subject or cell in need thereof.

[0127] Still further aspects relate to methods of treating a subject having a disease, disorder, or condition comprising administering the modified AAV disclosed herein to the subject. In some aspects, the disease, disorder, or condition is selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha- 1 -antitrypsin, amyotrophic lateral sclerosis, Alzheimer's, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesteremia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysarccharidosis (I-IX), hepatitis B, and hepatitis C. In one aspect, the hemophilia is characterized by one or more of factor VIII or factor IX deficiency. In some aspects, the muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery -Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and

oculopharyngeal muscular dystrophy.

[0128] In some aspects, guide RNA and/or the therapeutic polynucleotide is designed and/or selected to treat a disease, disorder, or condition selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha- 1 -antitrypsin, amyotrophic lateral sclerosis, Alzheimer's, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesteremia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysarccharidosis (I-IX), hepatitis B, and hepatitis C. In one aspect, the hemophilia is characterized by one or more of factor VIII or factor IX deficiency. In some aspects, the muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0129] In some aspects, the guide RNA and/or the therapeutic polynucleotide is designed and/or selected to target or repair a gene selected from the group of Factor VIII (F8, NM_000132, NM_019863), Factor IX (F9, NM_000133, NM_001313913), dystrophin (DMD, NM_000109, NM_004006, NM_004007, NM_004009, NM_004010), dysferlin (DYSF, NM_001 130455, NM_001130976, NM_001130977, NM_001130978,

NM_001130979), emerin (EMD, NM_0001 17), lamin A/C (LMNA, NM_001257374, NM_001282624, NM_001282625, NM_001282626, NM_005572), double homeobox 4 (DUX4, NM_001205218, NM_001278056, NM_001293798, NM_001306068), myotonin- protein kinase (MDPK, NM_001081560, NM_001081562, NM_001081563,

NM_001288764, NM_001288765), cellular nucleic acid-binding protein (CNBP,

NM_003418, NM_001127192, NM_001 127193, NM_001127194, NM_001 127195), polyadenylate-binding protein-2 (PABP-2, NM_004643), Alpha- 1 -antitrypsin, superoxide dismutase (SOD1 , NM_000454), alsin (ALS2, NM_001 135745, NM_020919), helicase senataxin (SETX, NM_015046), spatacsin (SPG11 , NM_001 160227, NM_025137), RNA- binding protein FUS/TLS (FUS, NM_001010850, NM_001170634, NM_001 170937, NM_004960), Vesicle-associated membrane protein-associated protein B/C (VAPB, NM_001195677, NM_004738), angiogenin (ANG, NM_001145, NM_001097577), TAR DNA-binding protein 43 (TARDBP, NM 007375), Polyphosphoinositide phosphatase (FIG4, NM_014845), optineurin (OPTN, M 001008211, NM_001008212, NM_001008213, NM_021980), ataxin-2 (ATXN2, NP_001297050, NP_001297052, NP_002964), valosin- containing protein (VCP, NM_007126), ubiquilin-2 (UBQLN2, NM_013444), sigma-1 receptor (SIGMAR1, NM_001282205, NM_001282206, NM_001282207, NM_001282208, NM_001282209), Charged multivesicular body protein 2b (CHMP2B, NM_001244644, NM_014043), profilin-1 (PFN1, NM_005022), Receptor tyrosine-protein kinase erbB-4 (ERBB4, NM_001042599, NM_005235), Heterogeneous nuclear ribonucleoprotein Al (HNRNPA1, NM_002136, NM_031157), matrin-3 (MATR3, NM_199189, NM_001194954, NM_001194955, NM_001194956, NM_001282278), tubulin alpha-4A chain (TUBA4A, NM_001278552, NM_006000), chromosome 9 open reading frame 72 (C9orf72,

NM_145005, NM_001256054, NM_018325), CHCDIO, SQSTMl (NM_001142298), TBKl, apolipoprotein E (NM_001302691, NM_000041, NM_001302688, NM_001302689, NM_001302690), SMN1 (NM_000344), SMN2 (NM_017411, NM_022875, NM_022876, NM_022877), CTFR (NM_000492), beta globin HBB PDB, CHM, alpha-synuclein (SNCA, NM_000345), parkin (PRKN, NM_004562), leucine-rich repeat kinase 2 (LRRK2 or dardarin, NM_198578), PTEN-induced putative kinase 1 (PINK1, NM_032409), DJ-1 (NM_001123377), acid maltase (NM_000152), UDP-glucuronosyltransferase 1

(NM_000463), PPT-1 (NM_000310), or ATP13A2 (NM_001141973).

[0130] Additional aspects of the invention relate to compositions comprising a carrier and the modified virus described in the embodiments disclosed herein.

[0131] Briefly, pharmaceutical compositions of the present invention may comprise a modified viral particle expressing Cas9 on its surface as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants;

chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, and/or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration. [0132] It is appreciated by those skilled in the art that gRNAs can be generated for target specificity to target a specific gene, optionally a gene associated with a disease, disorder, or condition. Thus, in combination with Cas9, the guide RNAs facilitate the target specificity of the CRISPR/Cas9 system. Further aspects such as promoter choice, as discussed above, may provide additional mechanisms of achieving target specificity - e.g., selecting a promoter for the guide RNA encoding polynucleotide that facilitates expression in a particular organ or tissue. Accordingly, the selection of suitable gRNAs for the pariticular disease, disorder, or condition is contemplated herein.

[0133] Administration of the modified AAV or compositions can be effected in one dose, continuously or intermittently throughout the course of treatment. Administration may be through any suitable mode of administration, including but not limited to: intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmuccosal, and inhalation.

[0134] Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy and the subject being treated. Single or multiple administrations can be carried out with the dose level and partem being selected by the treating physician. It is noted that dosage may be impacted by the route of administration. Suitable dosage formulations and methods of administering the agents are known in the art. Non-limiting examples of such suitable dosages may be as low as 1E+9 vector genomes to as much as 1E+17 vector genomes per administration.

[0135] In a further aspect, the modified viral particle and compositions of the invention can be administered in combination with other treatments, e.g. those approved treatments suitable for the particular disease, disorder, or condition. A non-limiting example includes the treatment of muscular dystrophy with a combination of the modified viral particle and one or more steroids.

[0136] This administration of the modified viral particle or compositions of the invention can be done to generate an animal model of the desired disease, disorder, or condition for experimental and screening assays. Modified AAV Capsids and Particles

[0137] The present disclosure provides also provides a specific embodiment, e.g., a modified adeno-associated virus (AAV) expressing Cas9 on its surface and methods of making the modified AAV and using the modified AAV. Adeno-associated virus (AAV) vectors are replication defective viruses that are engineered to deliver genetic cargo efficiently to cells. They are non-enveloped viruses that in their vector form only possess the inverted terminal repeats (ITR) of the original virus. The structural and enzymatic AAV proteins are supplied "in trans" by additional plasmids and are transfected together into a cell to generate the engineered particles for gene delivery. AAVs have been widely utilized for genetic therapy - and more specifically with CRISPR/Cas9 systems - due to their safety and efficiency. AAV efficiently infects a variety of cells and during the infection process the capsid binds to and enters the nucleus where the vector genome is delivered.

[0138] The AAV structural particle is composed of 60 protein molecules made up of VP1, VP2 and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins ordered into an icosahedral structure. It has been shown that AAV2 particles can support the insertion of peptides and proteins at various sites within the capsid structure. The ability to introduce unique peptides into the capsid has led to the development of AAV particles with altered tropism, which allows the virus to bind and infect cells and tissues that may normally be refractory to infection. In addition, large peptides and even functional proteins have been introduced into the capsid of AAV2 vectors with varying levels of success. A functional green fluorescent protein (GFP, 30 kD MW) containing AAV capsid was generated and produced infectious virus that was used to track cell infections.

[0139] One of the constraints with AAV vectors for gene delivery is the size limitation of the genetic insert that can be efficiently packaged into particles. For example, the size of the wild-type AAV2 genome is 4679 bases of single stranded DNA. Packaging even one of the new smaller variants of Cas9 {staphylococcus aureus Cas9, SaCas9, 130 kD MW) requires approximately 3255 bp just for the coding region. Adding a ubiquitous or tissue specific promoter to the construct may add another 500-800 bp. Include another 500 bp for a poly A addition sequence and the ITR's and the vector is close to the packaging capacity of an AAV particle. To achieve functional CRISPR/Cas9 gene correction a guide RNA ('"gRNA") with the target sequence must also be included. To have this RNA expressed further requires a minimal polIII promoter and termination sequence. Together these elements are too large to be combined into an AAV vector that is efficiently packaged. One can choose to package the Cas9 construct and guide RNA expression cassettes into separate vectors, but, for them to be functional, both viruses must infect the same target cells.

[0140] Rather than direct delivery, Applicant has generated plasmids to produce a modified AAV expressing Cas9 on its surface. During the normal course of AAV infection of a cell, the particle surface contains nuclear localization sequences, which direct the virus to traffic to the nucleus. Upon binding the nuclear pore complex the particle enters the nucleus and uncoats the vector genome. AAV capsid proteins are very stable inside the nucleus and can be found for many weeks after infection. By engineering an AAV vector to express the Cas9 enzyme on the surface of the virus particle one eliminates the need to package the Cas9 coding region within the particle and would allow the delivery of both a functional Cas9 enzyme and the guide RNA expression cassette within a single vector particle. These modified AAVs have been transfected or transduced into cells to demonstrate that the correct proteins are being produced. Applicant is producing research batches of the resulting virus for testing functional activity of these viruses in cell culture.

[0141] Aspects of the disclosure relate to a modified adeno-associated virus (AAV) expressing Cas9 on its surface comprising an AAV viral protein selected from the group of VP1, VP2, and VP3 fused to Cas9. In some embodiments, the AAV viral protein is VP2. In some embodiments, the Cas9 is S. aureus Cas9. In further embodiments, the Cas9 comprises the amino acid sequence provided in SEQ ID NO: 3 or an equivalent thereof. In some embodiments, the modified AAV comprises and/or encapsulates one or more guide RNAs or polynucleotides encoding said guide RNAs. It is appreciated by those skilled in the art that gRNAs can be generated for target specificity to target a specific gene, optionally a gene associated with a disease, disorder, or condition. Thus, in combination with Cas9, the guide RNAs facilitate the target specificity of the CRISPR/Cas9 system.

[0142] Further aspects of the disclosure relate to a recombinant expression system for the generation of such a modified AAV. In some embodiments the recombinant expression system comprises a plurality of plasmids; the plurality encoding all of the AAV viral proteins - VP1, VP2, and VP3. In some embodiments, each viral protein is encoded in a different plasmid. In some embodiments, one or more viral proteins is encoded in the same plasmid. In some embodiments, at least one viral protein is encoded as a fusion protein with Cas9.

[0143] Accordingly, embodiments disclosed herein relate to a recombinant expression system for the generation of a modified AAV expressing Cas9 on its surface comprising: (a) a plasmid comprising a DNA sequence encoding a fusion protein comprising Cas9 and an AAV viral protein selected from the group of VP1, VP2, and VP3, and (b) a plasmid comprising a DNA sequence encoding any AAV viral proteins selected from the group of VP1, VP2, and VP3 not comprised in the fusion protein of plasmid (a). In some

embodiments, the fusion protein comprises VP2. In some embodiments, the Cas9 is S.

aureus Cas9. In further embodiments, the Cas9 comprises the amino acid sequence provided in SEQ ID NO: 3. In some embodiments, plasmid (a) comprises a DNA sequence selected from the group of SEQ ID NO: 2 and SEQ ID NO: 5. In some embodiments, plasmid (b) comprises a DNA sequence selected from the group of SEQ ID NO: 1 and SEQ ID NO: 4. In some embodiments, the recombinant expression system further comprises a helper virus or helper plasmid. In some embodiments, the helper plasmid comprises the DNA sequence provided in SEQ ID NO: 6. In some embodiments, the recombinant expression further comprises a plasmid comprising a DNA sequence encoding one or more guide RNAs.

[0144] Some aspects relate to methods of producing the modified AAVs using the recombinant expression system disclosed herein. Aspects relate to a method of producing a modified AAV expressing Cas9 on its surface by transfecting one or more cells with the recombinant expression system disclosed herein. In some embodiments, the one or more cells are HEK293 cells.

[0145] Still further aspects relate to methods of treating a subject having a disease, disorder, or condition comprising administering the modified AAV disclosed herein to the subject. In some embodiments, the disease, disorder, or condition is selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha- 1 -antitrypsin, amyotrophic lateral sclerosis, Alzheimer's, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesteremia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysarccharidosis (I-IX), hepatitis B, and hepatitis C. In some embodiments, the hemophilia is characterized by one or more of factor VIII or factor IX deficiency. In some embodiments, the muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery -Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. Examples

[0146] The following examples are non-limiting and illustrative of procedures which can be used in various instances in carrying the disclosure into effect. Additionally, all references disclosed herein below are incorporated by reference in their entirety.

Example 1 - Generation of AAV particles with Cas9 surface expression

[0147] Applicant constructed two plasmids according to the schematic provided in FIG. 1. The sequences for these plasmids is provided as SEQ ID NO: 1, encoding the proteins for VPl (SEQ ID NO: 37) and VP3 (SEQ ID NO: 38), and SEQ ID NO: 2 or SEQ ID NO: 5, encoding the protein for a VP2-Cas9 fusion (SEQ ID NO: 36). Applicant constructed additional plasmids according to the schematic provided in FIGs. 2 and 9. The sequences for these plasmids is provided as SEQ ID NO: 4, encoding VPl AND VP3, SEQ ID NO: 2, encoding a VP2-Cas9 fusion, SEQ ID NO: 5, encoding a VP2-Cas9 fusion with an OLLAS epitope tag, SEQ ID NO: 6, encoding a helper plasmid, SEQ ID NO: 7, encoding a reporter (luciferase), and SEQ ID NO: 8, encoding a gRNA. Non limiting examples of VPl sequences include SEQ ID NO: 37, DNA base pairs numbered 5037 to 7253 of SEQ ID NO: 1, base pairs numbered 5037 to 7253 of SEQ ID NO: 4, and equivalents of each thereof. Nonlimiting examples of VP2 sequences include SEQ ID NO: 39, base pairs numbered 8786 to 10574 of SEQ ID NO: 5, and equivalents of each thereof. Nonlimiting examples of VP3 sequences include SEQ ID NO: 38, base pairs numbered 5646 to 7253 of SEQ ID NO: 1, base pairs numbered 5646 to 7253 of SEQ ID NO: 1, and an equivalent of each thereof. Nonlimiting examples of VP2-Cas9 fusion sequences include SEQ ID NO: 36, base pairs numbered 5532 to 1074 of SEQ ID NO: 5, base pairs numbered 5532 to 10565 of SEQ ID NO: 2, and equivalents of each thereof.

[0148] HEK293 cells are transfected with plasmids that encode for VPl + VP3 and in a separate plasmid, the Cas9-VP2 fusion protein (e.g., SEQ ID NO: 1 and SEQ ID NO: 2). In addition, the targeting vector containing the guide RNA sequence and, if needed, additional therapeutic polypeptide encoding a DNA repair template or other DNA sequence required for gene modification is also transfected or co-transfected into the HEK293 cells (e.g. SEQ ID NO: 8). An additional plasmid can be transfected or co-transfected that provides the viral helper function found in Adenovirus (E1A, E1B, E2A, E40RF6 and VA RNAs) or Herpes virus (among other viruses as well) to enable efficient AAV production. Alternatively, the AAV and Helper genes can be provided as separate plasmids or combined into multiples or a single plasmid if desired. Alternatively, the genes can be stably introduced into cells to generate stable packing cell lines. Alternatively, the genes can also be introduced into cells using viral vectors like baculo-virus or herpes virus to amplify and deliver large quantities of the needed genes to adherent or suspension grown cells.

[0149] For transfection, suspension adapted HEK293 cells are grown in serum free 293Expi media to a concentration of 5E+6 cells/mL. Cells are transfected with the plasmids as described above (e.g., pAAVrh74-Cas9-VP2, pAAVrh74-VPl-3, pHELP and scAAV-CMV- luc2Pv2 plasmids) using polyethylenimine (PEI) using standard transfection methods.

Briefly, the plasmid DNA is mixed with Opti-mem media separately and the PEI is mixed with Opti-mem separately. The diluted DNA and PEI mixes and combined, vortexed briefly and allowed to sit at room temperature for ten minutes for complex formation. The transfection mix is then added to the cells and the cells are incubated in shake flasks at 135 rpm and 37 degrees in a humidified incubator. Following transfection, the HEK293 cells are cultured to produce supernatant containing viral particles. Four days after transfection the virus is recovered from the media using depth filtration with a 0.45-micron filter (Millipore) and concentrated using a lOOkD MWCO spin concentrator (Pierce) and purified by iodixanol gradient (15-57%) ultracentrifugation (68,000 rpm, 18 degrees, 1 hour) and column chromatography (GE).

[0150] To identify expression of the intended proteins (e.g., VP1, VP3, and Cas9-VP2 fusion), a western blot is performed. A western blot is also performed to qualitatively analyze viral particles in rAAV fraction and final samples and to determine purity. Briefly, a BOLT SDS-Page gel method is performed. First, samples of viral supernatant are prepared in 1.5 mL epp tubes by adding 1 μΐ Bolt DTT reducing agent, 2.5 μΐ Bolt NuPAGE LDS 4X loading dye, and 6.5 μΐ of sample into each tube and pipette up and down to mix. Next, the samples are denatured by placing the tubes into a heat block set at 95 °C for 10 minutes. The Mini Gel Tank Electrophoresis system is assembled by placing the cassette into the tank and make sure the electrodes are on opposite sides. The comb and tape is removed the 10% Bis- Tris gel. The IX MOPS SDS running buffer by adding 20 mL of Bolt MOPS SDS 20X running buffer to 380 mL dH20. After heating the samples for 10 minutes, the tubes are cooled on ice for 1 minute and then centrifuge to get rid of any condensation. 10 μΐ of the denatured samples is added into each well. 10 μΐ of a standard ladder such as IX Mark 12 standard is added to the last well of the SDS-PAGE gel. The gel is run at 165 volts, 500mA (constant), for 45 minutes. The staining solution is prepared by adding 100 mL of 7.5% Acetic Acid and 10 μΐ SYPRO orange into a gel staining box and the gel is stained on a rocker set 60 rpm at room temperature for 1 hour. Once the gel is done shaking, the 7.5% Acetic Acid is replaced and the gel is stained with 75 mL of fresh 7.5% Acetic Acid for 5-10 minutes to wash residue from gel. An image of the gel is captured using an imaging system. Appropriate expression of the recombinant viral system is indicated by detecting bands that correspond to the predicted size of the expressed viral proteins. For example, VP1, VP2, and VP3 are approximately 87, 72, and 62 kDa respectively. saCas9 is approximately 127 kDa. The VP2-Cas9 fusion protein is approximately 193 kDa in size.

Example 2 - Treatment of Duchenne muscular dystrophy

[0151] Duchenne muscular dystrophy is an inherited X-linked recessive gene defect that affects approximately 1 in 5000 newborn males. The gene is 2.2 megabases (MB) in length and contains 79 exons. Truncated forms of the DMD gene have been tested as a gene replacement strategy but the truncated form does not provide full functionality. By developing methods to accurately correct the myriad of gene mutations that are specific in each individual a fully functional dystrophin gene can be restored for these patients.

[0152] The CRISPR/Cas9 system allows for simple replacement of the targeting sequence to provide specific gene correction. The disclosed AAV delivery system is used to efficiently target every major muscle with a single intravenous administration, and provides a robust therapeutic strategy to treat DMD.

[0153] A mouse model mdx is used to demonstrate the effectiveness of treating muscular dystrophy with the modified viral particle and the methods disclosed herein. Mdx mice bear a frame-disrupting mutation in the DMD gene which compromises the muscle myofibers and results in muscle deterioration. One strategy for potential gene repair is to remove at least one exon from the DMD gene, thus producing a truncated mRNA that is still in frame and produces a dystrophin protein that is at least partially functional. To directly edit the DMD gene in mice, a gene therapy approach with a modified AAV viral particle is used to deliver a guide RNA capable of excising exon 23 of the mouse DMD gene, concurrently with delivery a Cas9-viral capsid fusion protein. Because the AAV will ultimately be used to target skeletal muscle, an AAV with skeletal muscle tropism should be used such as AAV1, AAV6, AAV7, AAV8, or AAV9.

[0154] Modified Cas9 AAV particles are prepared as described above. Briefly, HEK293T cells are co-transfected with four plasmids. The first plasmid encodes AAV viral capsid proteins VPl and VP3 with VP2 deleted (e.g. SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4). The second plasmid encodes the saCas9-VP2 fusion protein (e.g. SEQ ID NO: 3). The third plasmid encodes viral assembly helper genes (e.g. SEQ ID NO: 6). The fourth plasmid encodes the guide RNA targeting the DMD gene under the control of a U6 promoter or another appropriate promoter for expression in the tissue of interest (e.g. SEQ ID NO: 8). Alternatively, a cell line in which necessary viral assembly genes such as the genes encoded in the first and/or third plasmids are stably introduced can be used in lieu of co-transfection with plasmids encoding those genes.

[0155] Methods for designing guide RNA sequences for targeting exon 23 of the mouse Dmd gene are known in the art. For example, see Tabebordbar, M. et al. (2016) Science 351(6271):407-411, incorporated herein by reference. Exemplary guide RNA target sequences appropriate for saCas9 cleavage of Dmd exon 23 are disclosed as SEQ ID NOs: 10-17. SEQ ID NOs: 10-17 target genomic sequences flanking exon 23, resulting of excision of exon 23. These sequences are cloned into the fourth plasmid, a scaffold guide RNA plasmid, to be packaged into the assembled modified viral particle. Control guide RNAs are also prepared that do not target the Dmd gene.

[0156] Following co-transfection, assembled modified viral particles are harvested and tested for saCas9-VP2 protein expression, as well as expression of VPl and VP3 by westem blot as described in Example 1. The packaged virus is also assayed for viral titer which should range from about 10^Λ8 GC/mL to 10^Λ17 GC/mL, with titer optimally of about 10^Λ13 GC/mL. Viral titer can be assayed by westem blot or by viral genome copy number by qPCR and compared to copy number standard samples. Following confirmation of fusion protein expression and sufficient viral titer, the modified viral particles are administered ex vivo to cells harvested from mdx mice to confirm efficient excision of exon 23. The harvested cells from the mdx mice (e.g., muscle cells, muscle stem cells, liver cells, fibroblasts, adipose stem cells, or any other cells compatible with the AAV serotype used) bear the genomic Dmd mutation. Upon transduction with the modified viral particles, they can be assayed for efficient exon 23 excision by PCR using primers that span the deletion region. Efficient operation of the CRISPR system can be measured by comparing the relative levels of PCR products of primers that span exon 23, primers within exon 23, and products where one primer is outside the deleted region and the other is inside the deleted region of exon 23. Efficient excision will be demonstrated the primers spanning exon 23 produce the most abundant product. Additional confirmation of efficient CRISPR activity can be ascertained by western blot for the repaired Dystrophin protein product.

[0157] Following confirmation of the CRISPR system's efficient excision, the modified viral particles can be administered ex vivo or in vitro to muscle stem or progenitor cells from the mdx mice such as satellite cells. Upon exon 23 excision, the CRISPR modified cells are transplanted back into the mice via intramuscular injection. Effectiveness of cell therapy with the cells treated with modified AAV is measured by improved muscle morphology, decreases in sarcolemmal localization of the multimeric dystrophin-glycoprotein complex and neuronal nitric-oxide synthase, as well as detection of Dystrophin expression.

[0158] Alternatively, the modified viral particles can be administered in vivo to muscle tissue through localized tissue injection such as intramuscular injection, intraperitoneal injection, systemic injection, or by tail vein injection. Effectiveness of viral gene therapy with the modified saCas9 AAV is measured by improved muscle morphology, decreases in sarcolemmal localization of the multimeric dystrophin-glycoprotein complex and neuronal nitric-oxide synthase, as well as detection of Dystrophin expression.

[0159] To treat muscular dystrophy in humans, guide RNAs are designed that target one or more of the following genes that cause muscular dystrophy: dystrophin (DMD, NM_000109, NM_004006, NM_004007, NM_004009, NM_004010), dysferlin (DYSF, NM_001130455, NM_001130976, NM_001130977, NM_001130978, NM_001130979), emerin (EMD, NM_000117), lamin A/C (LMNA, NM_001257374, NM_001282624, NM_001282625, NM_001282626, NM_005572), double homeobox 4 (DUX4, NM_001205218,

NM_001278056, NM_001293798, NM_001306068), myotonin-protein kinase (MDPK, NM_001081560, NM_001081562, NM_001081563, NM_001288764, NM_001288765), cellular nucleic acid-binding protein (CNBP, NM_003418, NM_001127192,

NM_001127193, NM_001127194, NM_001127195), polyadenylate-binding protein-2 (PABP-2, NM_004643). The guide RNA is designed to direct Cas9 to excise an exon via non-homologous end joining (NHEJ) causing an in frame truncation product that produces a functional protein product. Alternatively, the guide RNA can be designed to repair a gene via homology directed repair. This method uses a therapeutic DNA encoding a wild-type DNA sequence or replacement sequence to be used as a template for repair of the cleaved region.

[0160] Modified viral particles with exterior Cas9 and encapsulating a polynucleotide comprising the guide RNA, and, optionally, the therapeutic template DNA, are prepared as described above. Viral protein expression and titer are assayed by western blot and PCR as described above. Efficiency of CRISPR -mediated gene editing is assayed by designing PCR primers that detect the repaired DNA fragment. Viral particles are administered to muscle tissue via intramuscular injection or systemic delivery. Expression of repaired gene product can be detected by PCR, histological staining, or western blot of treated muscle tissue.

Example 3 - Treatment of Hemophilia

[0161] To treat hemophilia, guide RNAs are designed to direct CRISPR-mediated gene repair to Factor VIII (F8, NM_000132, NM_019863) or Factor IX (F9, NM_000133, NM_001313913). Additionally, therapeutic polynucleotides are prepared to provide templates for repair of Factor VIII (F8, NM_000132, NM_019863) or Factor IX (F9, NM_000133, NM_001313913). Modified viral particles with exterior Cas9 and encapsulating a polynucleotide comprising the guide RNA and the therapeutic template DNA, are prepared as described above. Viral protein expression and titer are assayed by western blot and PCR as described above. Efficiency of CRISPR -mediated gene editing is determined by designing PCR primers that detect the repaired DNA fragment. In one aspect, modified viral particles are administered to stem cells, hepatocyte precursor cells, or hepatocytes to correct the factor VIII or IX genes. Alternatively, modified viral particles are administered directly to a subject with hemophilia by injection directly into the liver or by systemic delivery. Successful gene repair is detected by detecting functional Factor VIII or Factor IX protein within the treated cell or subject with hemophilia.

Equivalents

[0162] It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and

modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0163] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0164] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

References

The following articles are referenced in the disclosure hereinabove and are incorporated by reference, in their entirety:

1 Wu, P. et al. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism. J Virol 74, 8635-8647 (2000).

2 Loiler, S. A. et al. Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver. Gene Ther 10, 1551-1558,

doi: 10.1038/sj.gt.3302046 (2003).

3 Warrington, K. H., Jr. et al. Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol 78, 6595-6609, doi: 10.1128/JVI.78.12.6595-6609.2004 (2004).

4 Tenney, R. M., Bell, C. L. & Wilson, J. M. AAV 8 capsid variable regions at the twofold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating. Virology 454-455, 227-236, doi: 10.1016/j.virol.2014.02.017 (2014).

5 Nicolson, S. C. & Samulski, R. J. Recombinant adeno-associated virus utilizes host cell nuclear import machinery to enter the nucleus. J Virol 88, 4132-4144,

doi: 10.1128/JVI.02660-13 (2014).

6 Ran, F. A. Adaptation of CRISPR nucleases for eukaryotic applications, Analytical Biochemistry. http://dx.doi.Org/10.1016/j.ab.2016.10.018 (2016).

7 Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184-1188, doi: 10.1126/science.1254445 (2014).

8 Bengtsson, N. E. et al. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat Commun 8, 14454, doi: 10.1038/ncommsl4454 (2017). SEQUENCE LISTING

[0165] A description of the non-limiting exemplary vectors and the sequences thereof discussed herein is provided herein below:

pNL-Rep2-Caprh74-AVB-VPl-3

LOCUS pNLRep2-Caprh74- 10538 bp DNA circular SYN 23-MAR-2016 DEFINITION Knocks out VP2 expression, 5448 A-G

ACCESSION pNLRep2-Caprh74- ORGANISM Unknown

REFERENCE 1 (bases 1 to 10538)

COMMENT SECID/File created by Clone Manager, Scientific & Educational Software COMMENT SECNOTES|GenBank 10538 bp DNA circular 20-MAR-2015 FEATURES Location/Qualifiers

misc_feature 84..815

/note="Rep78 5"'

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="Rep78"

/SECDescr="Rep78 5"'

misc_feature 756..815

/note="Rep52 5"'

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="Rep52"

/SECDescr="Rep52 5"'

misc_feature 816..3886

/note="Human Collagen Intron"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="H Coll Int"

/SECDescr="Human Collagen Intron"

misc_feature 3887..5017

/note="Rep52 3"'

/SECDrawAs="Gene"

/SECStyleId=l /SECName="Rep52" /SECDescr="Rep52 3"' misc_feature 3887..5017

/note="Rep78 3"'

/SECDrawAs="Gene" /SECStyleId=l

/SECName="Rep78" /SECDescr="Rep78 3"' misc_feature 4741..4742

/note="splice donor" /SECDrawAs="Region" /SECStyleId=l

/SECName="SD"

/SECDescr="splice donor" misc_feature 4741..5061

/note="Rep INTRON" /SECDrawAs="Region" /SECStyleId=l

/SECName="Rep int" /SECDescr="Rep INTRON" misc_feature 5033..5034

/note="splice acceptor" /SECDrawAs="Region" /SECStyleId=l

/SECName="SA"

/SECDescr="splice acceptor"

CDS 5037..7253

/gene="VPl "

/SECDrawAs="Gene" /SECStyleId=l

/SECName="VP l " misc_feature 5060..5061

/note- ' splice acceptor" /SECDrawAs="Region" /SECStyleId=l

/SECName="SA"

/SECDescr- 'splice acceptor"

misc_feature 5062..5086

/note="REP68/40 3' end AAV2 wt is RLARGHSL with rh.74

capsid it is RLARGQPL ! "

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="REP68/40"

/SECDescr="REP68/40 3' end AAV2 wt is RLARGHSL with rh.74 capsid it is RLARGQPL ! "

CDS 5646.7253

/gene="VP3"

/SECDrawAs="Gene"

/SECStyleId=l

misc_feature complement(7254..7411)

/note="3' UTR"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="3"

/SECDescr="3' UTR"

misc_feature 7428..7507

/note="p5 Promoter"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="p5"

/SECDescr="p5 Promoter"

CDS complement(8893..9753)

/gene="amp"

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="amp"

ORIGIN (SEQ ID NO: 1)

1 cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg 61 ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca

121 gcgaccttga cgagcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga

181 aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc

241 tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg

301 ccccggaggc tcttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg

361 tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc

421 gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg

481 cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca

541 tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg

601 aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc

661 tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg

721 cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg

781 acaaggggat tacctcggag aagcagtgga tccaggtgag taattgacaa agccaaacac

841 caccatttgc cgagcacttt agagtttaca ggtttgtttc tcttgaccct caaaacaaac

901 ctgtgaggca tagggagtat tgctatccct taagaattca cccccagtgt gcccatcaaa

961 acctcccagg ctgagtctgc acagttgaag gaggaaggat aggaatggga gggtcgatgg

1021 gtgaaagcat gattctctta accagtccag attatcaggt aatcccttca acaaccacca

1081 cccactccct gggcaatcca gctggagttt acagacagac ttagctggct atagcaccac

1141 cgtgctactc tctgttcttc ctggttgctc aaatgcccta gaaaagtgga acaggtgagc

1201 atcaactcac agggctctat gctggctgct gctgcgaggg atgttatgct atagtaccag

1261 gggccaccat tccataggca cttcctgtgt ttaataccct atatgcttta cttcatctca

1321 tcttcctcca tatcctgaga ggtggttcta ttcttctccc cattttacgg atg aaaaaac

1381 cgagacacag aaaggtgaaa tagcttaaga taaatggtgc cttgcagcct tagactctgg

1441 tggcctctag ttaatgtggg aaattaaggg tgaggggatt ggcagctgat ggagggtgca

1501 gggtgccaga cagaggcgtt tagctctgat cccttagcaa tagagagtcc ttgtaggcac

1561 ttggtcaggc gagtgatgcg atgaaagctg tgtttaagaa agattatgct ttctgctgat

1621 ttcatacccc caacacccaa gctctgaggc ccctcctcac aggtccttgc agggctggcc

1681 aaaataaagc agcttcactc cgttgtgctg ctttccagct aatgtgtctg tttggcagaa

1741 gtttccctca aaggcagatc agtgaaataa gcagaagcct cgacccccct ttgtcagcca

1801 gagctgctga agtgccttgc cccagggtca ctttgtgtga ggggattaga gagcactggg

1861 gctgccaaga aacactgccg tttctacaga ttagcaggac gctggcttgt ggccttctag

1921 cgaggctcag agctgcggtg gccctagtct gcatgggcta aagacaagct ccatctcctg

1981 tccttgttcc ctccttcctg ggcacagccg ccctgcttct tggttctctc tgttggttcc

2041 tgtccgcacg gtagttaggc tggcagcgtg tgtaggattt ggcttagaag attgacaaca 2101 ttgcctttga gcccttcttt gctactcctc cctctcccct cccatcagac tcctctctgg

2161 agtctgctct gcgaggcctc tgctctgtgg tatcccagca gccttctcag ccttgacttc

2221 cagaaggggg ctgtgcagtg tccggggtgt gcaggcccca gacacggggt aggctcatgg

2281 agatccaagt gctgatctag tgtcaaggct ggcctggaga ctgggctggg ttggtgtctg

2341 cctgctgtgg tcatgtgccc tcccttgggc ctgtatcctc tctccagact tgctgcaggg

2401 agaggtggca gatgtcagcc tagttctggc ctctcagagc agcatggcag ctccctttca

2461 ctcaggccca ggctgggccc tcctgctggc tgacccctgg ggagagggtg ctccagagct

2521 ccccaaggaa cagcttcccg aagcagccag gccagcccag aggggctgtg gccaatcctg

2581 aagctttatg ttcctgctga cattttttct aagttttctc ttgctttcct cttaaatgcc

2641 aatctggaga gtctccgtta ggagaaatgg accccagcca ggaagaagag ttgagttgta

2701 tttaaaacac gagctccccc taaagcatcc ttctttagct tctaaggaga ggcagagact

2761 gacaggcagg actcagcagg aaaaggtacc cccctgacct gctcagtcag gccctaggcc

2821 cagctccacc cagcctgtgg cccccagagt ttcggtaaag agttccctgg gccttaagga

2881 accttgagag agcatttgag gggtgccacc acaaacttgg cagaaaaaac cctccccctc

2941 caagtccagt cctagagaag gagctggcaa ccttgccttg ctttgtaagc aaaagcctct

3001 tagggcttga gctcagatgt agtgtttgag ctgtggctgg tgccctgccc catcagggag

3061 ccaatggtag acatcctatg ggcatctttg ttttccgtaa gagcaggctg tctggggatg

3121 ggccagagga agaggcgacc tggagtcaac caagaggagg ccttaaccaa gccttaacca

3181 cagaggttaa ccaagccttg aaagcgcttc cccctgagca ggcaggaagc actgagtcca

3241 catggttgcc tcgctgtttc atttccttac actcaattct ctcagtcttt aaatgatcac

3301 ttggccttga agttacggat atttggggtc tgaactgaag ttgaagaaaa gaggaaatga

3361 tttaagcttt gtttaagatt aggggccagg tgcggtggct cacgcctgta atcccagcac

3421 cttgggagcc tgaggcgggt ggatcacctg aggtcaggag ttccagacca gcctggccaa

3481 catagcaaaa cccagtctct actaaaaata acaataaaaa aattagccag gtgtggtgac

3541 acatgcctgt aatcccagtt actcaggagg ctgaggcaga attgcttgaa cttgagaggt

3601 ggaggttgta gtgagccaag accgcaccac tgcactccag cctggcgaca gagccagact

3661 ccgtctcaaa aacaacaaca aaaaagatta gaagaagccc attactgcct tctggccacc

3721 cactcgcaca gacaccaaaa ctgcagccca cacctcgcca tcctcgtgct ctgccctggg

3781 acaccccagg cacagtgtgt ccttcgtttt ctgtaagggt gggctgggag cagggacgga

3841 cagggcctgt gggcacctct catggtcact tccttcttgc tcacaggagg accaggcctc

3901 atacatctcc ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa

3961 tgcgggaaag attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc

4021 cgtggaggac atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc

4081 ccaatatgcg gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac 4141 catctggctg tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca

4201 cactgtgccc ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg

4261 tgtcgacaag atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc

4321 ggccaaagcc attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc

4381 ccagatagac ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga

4441 cgggaactca acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga

4501 actcacccgc cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt

4561 tttccggtgg gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg

4621 tggagccaag aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg

4681 cgagtcagtt gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag

4741 gtaccaaaac aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca

4801 atgcgagaga atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt

4861 agagtgcttt cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa

4921 actgtgctac attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct

4981 ggtcaatgtg gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtatgg

5041 ctgccgatgg ttatcttcca gattggctcg aggacaacct ctctgagggc attcgcgagt

5101 ggtgggacct gaaacctgga gccccgaaac ccaaagccaa ccagcaaaag caggacaacg

5161 gccggggtct ggtgcttcct ggctacaagt acctcggacc cttcaacgga ctcgacaagg

5221 gggagcccgt caacgcggcg gacgcagcgg ccctcgagca cgacaaggcc tacgaccagc

5281 agctccaagc gggtgacaat ccgtacctgc ggtataatca cgccgacgcc gagtttcagg

5341 agcgtctgca agaagatacg tcttttgggg gcaacctcgg gcgcgcagtc ttccaggcca

5401 aaaagcgggt tctcgaacct ctgggcctgg ttgaatcgcc ggttaaggcg gctcctggaa

5461 agaagagacc ggtagagcca tcaccccagc gctctccaga ctcctctacg ggcatcggca

5521 agaaaggcca gcagcccgca aaaaagagac tcaattttgg gcagactggc gactcagagt

5581 cagtccccga ccctcaacca atcggagaac caccagcagg cccctctggt ctgggatctg

5641 gtacaatggc tgcaggcggt ggcgctccaa tggcagacaa taacgaaggc gccgacggag

5701 tgggtagttc ctcaggaaat tggcattgcg attccacatg gctgggcgac agagtcatca

5761 ccaccagcac ccgcacctgg gccctgccca cctacaacaa ccacctctac aagcaaatct

5821 ccaacgggac ctcgggagga agcaccaacg acaacaccta cttcggctac agcaccccct

5881 gggggtattt tgacttcaac agattccact gccacttttc accacgtgac tggcagcgac

5941 tcatcaacaa caactgggga ttccggccca agaggctcaa cttcaagctc ttcaacatcc

6001 aagtcaagga ggtcacgcag aatgaaggca ccaagaccat cgccaataac cttaccagca

6061 cgattcaggt ctttacggac tcggaatacc agctcccgta cgtgctcggc tcggcgcacc

6121 agggctgcct gcctccgttc ccggcggacg tcttcatgat tcctcagtac gggtacctga 6181 ctctgaacaa tggcagtcag gctgtgggcc ggtcgtcctt ctactgcctg gagtactttc

6241 cttctcaaat gctgagaacg ggcaacaact ttgaattcag ctacaacttc gaggacgtgc

6301 ccttccacag cagctacgcg cacagccaga gcctggaccg gctgatgaac cctctcatcg

6361 accagtactt gtactacctg tcccggactc aaagcacggg cggtactgca ggaactcagc

6421 agttgctatt ttctcaggcc gggcctaaca acatgtcggc tcaggccaag aactggctac

6481 ccggtccctg ctaccggcag caacgcgtct ccacgacact gtcgcagaac aacaacagca

6541 actttgcctg gacgggtgcc accaagtatc atctgaatgg cagagactct ctggtgaatc

6601 ctggcgttgc catggctacc cacaaggacg acgaagagcg attttttcca tccagcggag

6661 tcttaatgtt tgggaaacag ggagctggaa aagacaacgt ggactatagc agcgtgatgc

6721 taaccagcga ggaagaaata aagaccacca acccagtggc cacagaacag tacggcgtgg

6781 tggccgataa cctgcaacag caaaacgccg ctcctattgt aggggccgtc aatagtcaag

6841 gagccttacc tggcatggtg tggcagaacc gggacgtgta cctgcagggt cccatctggg

6901 ccaagattcc tcatacggac ggcaactttc atccctcgcc gctgatggga ggctttggac

6961 tgaagcatcc gcctcctcag atcctgatta aaaacacacc tgttcccgcg gatcctccga

7021 ccaccttcag ccaggccaag ctggcttctt tcatcacgca gtacagtacc ggccaggtca

7081 gcgtggagat cgagtgggag ctgcagaagg agaacagcaa acgctggaac ccagagattc

7141 agtacacttc caactactac aaatctacaa atgtggactt tgctgtcaat actgagggta

7201 cttattccga gcctcgcccc attggcaccc gttacctcac ccgtaatctg taattacatg

7261 ttaatcaata aaccggttaa ttcgtttcag ttgaactttg gtctcctgtc cttcttatct

7321 tatcggttac catagaaact ggttacttat taactgcttg gtgcgcttcg cgataaaaga

7381 cttacgtcat cgggttaccc ctagtgatgg agcggccgct ttcagttgaa ctttggtctc

7441 tgcgtatttc tttcttatct agtttccatg ctctagaggt cctgtattag aggtcacgtg

7501 agtgttttgc gacattttgc gacaccatgt ggtcacgctg ggtatttaag cccgagtgag

7561 cacgcagggt ctccattttg aagcgggagg tttgaacgcg cagccgccaa gccgaattct

7621 gcagatatcc atcacactgg cggccgctcg actagagcgg ccgccaccgc ggtggagctc

7681 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct

7741 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat

7801 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc

7861 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg

7921 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct

7981 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt

8041 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc

8101 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga

8161 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 8221 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac

8281 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg

8341 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc

8401 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag

8461 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt

8521 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt

8581 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg

8641 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac

8701 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca

8761 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac

8821 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac

8881 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt

8941 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt

9001 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt

9061 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc

9121 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa

9181 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg

9241 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt

9301 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc

9361 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt

9421 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg

9481 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac

9541 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc

9601 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt

9661 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg

9721 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag

9781 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa

9841 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat

9901 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc

9961 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt

10021 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa

10081 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg

10141 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga

10201 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 10261 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 10321 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 10381 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 10441 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag

10501 cgcgcgtaat acgactcact atagggcgaa ttgggtac

pNL-Rep2-Caprh74-AVB-VPl-3

LOCUS pNLRep2-Caprh74- 13850 bp DNA circular SYN 23-MAR-2016 DEFINITION Ligation of pX601-AAV-CMV~N 696 to 4011 product cut Nhel..6 to

NsiL.3331 into pNLRep2-Caprh74-AVB-VP2-NN cut NsiL.5464 to

NheL.5451

ACCESSION pNLRep2-Caprh74- ORGANISM Unknown

REFERENCE 1 (bases 1 to 13850)

COMMENT SECNOTES I Vector molecule: pNLRep2-Caprh74-AVB-VP2-NN cut NsiL.5464 to NheL.5451

Fragment ends: Nsil and Nhel

Fragment size: 10525

Insert molecule: pX601-AAV-CMV~N 696 to 4011 product cut Nhel..6 to NsiL.3331

Fragment ends: Nhel and Nsil

Fragment size: 3325

FEATURES Location/Qualifiers

misc_feature 84..815

/note="Rep68 5"'

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="Rep68"

/SECDescr="Rep68 5"'

misc_feature 84..815

/note="Rep78 5"'

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="Rep78"

/SECDescr="Rep78 5"' misc_feature 756..815

/note="Rep40 5"'

/SECDrawAs="Gene" /SECStyleId=l

/SECName="Rep40" /SECDescr="Rep40 5"' misc_feature 756..815

/note="Rep52 5"'

/SECDrawAs="Gene" /SECStyleId=l

/SECName="Rep52" /SECDescr="Rep52 5"' misc_feature 816..3886

/note="Human Collagen Intron /SECDrawAs="Region" /SECStyleId=l

/SECName="H Coll Intron" /SECDescr="Human Collagen misc_feature 3887..5017

/note="Rep52 3"'

/SEC Draw As=" Gene" /SECStyleId=l

/SECName="Rep52"

/SECDescr="Rep52 3'" misc_feature 3887..5017

/note="Rep78 3"'

/SEC Draw As=" Gene" /SECStyleId=l

/SECName="Rep78"

/SECDescr="Rep78 3'" misc_feature 4741..4742

/note="splice donor"

/SECDrawAs="Region"

/SECStyleId=l /SECName="SD"

/SECDescr="splice donor"

misc_feature 4741..5061

/note="Rep INTRON"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="Rep intron"

/SECDescr="Rep INTRON"

misc_feature 5033..5034

/note=" splice acceptor"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="SA"

/SECDescr="splice acceptor"

CDS 5037..10565

/gene="VP2-Cas9"

/product="fusion protein"

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="VP2-Cas9"

/SECDescr="fusion protein"

misc_feature 5060..5061

/note=" splice acceptor"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="SA"

/SECDescr="splice acceptor"

misc_feature 5062..5086

/note="REP68/40 3' end AAV2 wt is RLARGHSL with rh.74 capsid it is RLARGQPL ! "

/SECDrawAs="Gene"

/SECStyleId=l

/SECName="REP68" /SECDescr="REP68/40 3' end AAV2 wt is RLARGHSL with rh.74 capsid it is RLARGQPL ! "

misc_feature 5084..5086

/note="Rep 68/40 stop"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="Rep"

/SECDescr="Rep 68/40 stop"

CDS 5457..8772

/gene="'saCas9"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="saCas9"

misc_feature 8730..8771

/gene="OLLAS"

/product="epitope tag"

/SECDrawAs="Region"

/SECStyleId=l

misc_feature complement(l 0566..10723)

/note="3' UTR"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="3"

/SECDescr="3' UTR"

misc_feature 10740..10819

/note="p5 Promoter"

/SECDrawAs="Region"

/SECStyleId=l

/SECName="p5"

/SECDescr="p5 Promoter"

CDS complement 12205..13065)

/gene="amp"

/SECDrawAs="Gene"

/SECStyleId=l /SECName="amp"

ORIGIN (SEQ ID NO: 2)

1 cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg

61 ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca

121 gcgaccttga cgagcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga

181 aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc

241 tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg

301 ccccggaggc tcttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg

361 tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc

421 gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg

481 cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca

541 tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg

601 aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc

661 tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg

721 cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg

781 acaaggggat tacctcggag aagcagtgga tccaggtgag taattgacaa agccaaacac

841 caccatttgc cgagcacttt agagtttaca ggtttgtttc tcttgaccct caaaacaaac

901 ctgtgaggca tagggagtat tgctatccct taagaattca cccccagtgt gcccatcaaa

961 acctcccagg ctgagtctgc acagttgaag gaggaaggat aggaatggga gggtcgatgg

1021 gtgaaagcat gattctctta accagtccag attatcaggt aatcccttca acaaccacca

1081 cccactccct gggcaatcca gctggagttt acagacagac ttagctggct atagcaccac

1141 cgtgctactc tctgttcttc ctggttgctc aaatgcccta gaaaagtgga acaggtgagc

1201 atcaactcac agggctctat gctggctgct gctgcgaggg atgttatgct atagtaccag

1261 gggccaccat tccataggca cttcctgtgt ttaataccct atatgcttta cttcatctca

1321 tcttcctcca tatcctgaga ggtggttcta ttcttctccc cattttacgg atg aaaaaac

1381 cgagacacag aaaggtgaaa tagcttaaga taaatggtgc cttgcagcct tagactctgg

1441 tggcctctag ttaatgtggg aaattaaggg tgaggggatt ggcagctgat ggagggtgca

1501 gggtgccaga cagaggcgtt tagctctgat cccttagcaa tagagagtcc ttgtaggcac

1561 ttggtcaggc gagtgatgcg atgaaagctg tgtttaagaa agattatgct ttctgctgat

1621 ttcatacccc caacacccaa gctctgaggc ccctcctcac aggtccttgc agggctggcc

1681 aaaataaagc agcttcactc cgttgtgctg ctttccagct aatgtgtctg tttggcagaa

1741 gtttccctca aaggcagatc agtgaaataa gcagaagcct cgacccccct ttgtcagcca

1801 gagctgctga agtgccttgc cccagggtca ctttgtgtga ggggattaga gagcactggg

1861 gctgccaaga aacactgccg tttctacaga ttagcaggac gctggcttgt ggccttctag 1921 cgaggctcag agctgcggtg gccctagtct gcatgggcta aagacaagct ccatctcctg

1981 tccttgttcc ctccttcctg ggcacagccg ccctgcttct tggttctctc tgttggttcc

2041 tgtccgcacg gtagttaggc tggcagcgtg tgtaggattt ggcttagaag attgacaaca

2101 ttgcctttga gcccttcttt gctactcctc cctctcccct cccatcagac tcctctctgg

2161 agtctgctct gcgaggcctc tgctctgtgg tatcccagca gccttctcag ccttgacttc

2221 cagaaggggg ctgtgcagtg tccggggtgt gcaggcccca gacacggggt aggctcatgg

2281 agatccaagt gctgatctag tgtcaaggct ggcctggaga ctgggctggg ttggtgtctg

2341 cctgctgtgg tcatgtgccc tcccttgggc ctgtatcctc tctccagact tgctgcaggg

2401 agaggtggca gatgtcagcc tagttctggc ctctcagagc agcatggcag ctccctttca

2461 ctcaggccca ggctgggccc tcctgctggc tgacccctgg ggagagggtg ctccagagct

2521 ccccaaggaa cagcttcccg aagcagccag gccagcccag aggggctgtg gccaatcctg

2581 aagctttatg ttcctgctga cattttttct aagttttctc ttgctttcct cttaaatgcc

2641 aatctggaga gtctccgtta ggagaaatgg accccagcca ggaagaagag ttgagttgta

2701 tttaaaacac gagctccccc taaagcatcc ttctttagct tctaaggaga ggcagagact

2761 gacaggcagg actcagcagg aaaaggtacc cccctgacct gctcagtcag gccctaggcc

2821 cagctccacc cagcctgtgg cccccagagt ttcggtaaag agttccctgg gccttaagga

2881 accttgagag agcatttgag gggtgccacc acaaacttgg cagaaaaaac cctccccctc

2941 caagtccagt cctagagaag gagctggcaa ccttgccttg ctttgtaagc aaaagcctct

3001 tagggcttga gctcagatgt agtgtttgag ctgtggctgg tgccctgccc catcagggag

3061 ccaatggtag acatcctatg ggcatctttg ttttccgtaa gagcaggctg tctggggatg

3121 ggccagagga agaggcgacc tggagtcaac caagaggagg ccttaaccaa gccttaacca

3181 cagaggttaa ccaagccttg aaagcgcttc cccctgagca ggcaggaagc actgagtcca

3241 catggttgcc tcgctgtttc atttccttac actcaattct ctcagtcttt aaatgatcac

3301 ttggccttga agttacggat atttggggtc tgaactgaag ttgaagaaaa gaggaaatga

3361 tttaagcttt gtttaagatt aggggccagg tgcggtggct cacgcctgta atcccagcac

3421 cttgggagcc tgaggcgggt ggatcacctg aggtcaggag ttccagacca gcctggccaa

3481 catagcaaaa cccagtctct actaaaaata acaataaaaa aattagccag gtgtggtgac

3541 acatgcctgt aatcccagtt actcaggagg ctgaggcaga attgcttgaa cttgagaggt

3601 ggaggttgta gtgagccaag accgcaccac tgcactccag cctggcgaca gagccagact

3661 ccgtctcaaa aacaacaaca aaaaagatta gaagaagccc attactgcct tctggccacc

3721 cactcgcaca gacaccaaaa ctgcagccca cacctcgcca tcctcgtgct ctgccctggg

3781 acaccccagg cacagtgtgt ccttcgtttt ctgtaagggt gggctgggag cagggacgga

3841 cagggcctgt gggcacctct catggtcact tccttcttgc tcacaggagg accaggcctc

3901 atacatctcc ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa 3961 tgcgggaaag attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc

4021 cgtggaggac atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc

4081 ccaatatgcg gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac

4141 catctggctg tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca

4201 cactgtgccc ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg

4261 tgtcgacaag atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc

4321 ggccaaagcc attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc

4381 ccagatagac ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga

4441 cgggaactca acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga

4501 actcacccgc cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt

4561 tttccggtgg gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg

4621 tggagccaag aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg

4681 cgagtcagtt gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag

4741 gtaccaaaac aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca

4801 atgcgagaga atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt

4861 agagtgcttt cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa

4921 actgtgctac attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct

4981 ggtcaatgtg gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtatgg

5041 ctgccgatgg ttatcttcca gattggctcg aggacaacct ctctgagggc attcgcgagt

5101 ggtgggacct gaaacctgga gccccgaaac ccaaagccaa ccagcaaaag caggacaacg

5161 gccggggtct ggtgcttcct ggctacaagt acctcggacc cttcaacgga ctcgacaagg

5221 gggagcccgt caacgcggcg gacgcagcgg ccctcgagca cgacaaggcc tacgaccagc

5281 agctccaagc gggtgacaat ccgtacctgc ggtataatca cgccgacgcc gagtttcagg

5341 agcgtctgca agaagatacg tcttttgggg gcaacctcgg gcgcgcagtc ttccaggcca

5401 aaaagcgggt tctcgaacct ctgggcctgg ttgaatcgcc ggttaagatg gctagcggcg

5461 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact accggtgcca

5521 ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccaagcgga

5581 actacatcct gggcctggac atcggcatca ccagcgtggg ctacggcatc atcgactacg

5641 agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca

5701 acgagggcag gcggagcaag agaggcgcca gaaggctgaa gcggcggagg cggcatagaa

5761 tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga

5821 gcggcatcaa cccctacgag gccagagtga agggcctgag ccagaagctg agcgaggaag

5881 agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg

5941 tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg 6001 ccctggaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag

6061 tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc

6121 tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc tacatcgacc

6181 tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga

6241 aggacatcaa agaatggtac gagatgctga tgggccactg cacctacttc cccgaggaac

6301 tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca

6361 atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca

6421 tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc

6481 tcgtgaacga agaggatatt aagggctaca gagtgaccag caccggcaag cccgagttca

6541 ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga

6601 acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca

6661 tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagatc gagcagatct

6721 ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccatc aacctgatcc

6781 tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg

6841 tgcccaagaa ggtggacctg tcccagcaga aagagatccc caccaccctg gtggacgact

6901 tcatcctgag ccccgtcgtg aagagaagct tcatccagag catcaaagtg atcaacgcca

6961 tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact

7021 ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag accaacgagc

7081 ggatcgagga aatcatccgg accaccggca aagagaacgc caagtacctg atcgagaaga

7141 tcaagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg

7201 aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt

7261 ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac agcaagaagg

7321 gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct

7381 tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag

7441 agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc

7501 ggaacctggt ggataccaga tacgccacca gaggcctgat gaacctgctg cggagctact

7561 tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctttc

7621 tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg

7681 acgccctgat cattgccaac gccgatttca tcttcaaaga gtggaagaaa ctggacaagg

7741 ccaaaaaagt gatggaaaac cagatgttcg aggaaaagca ggccgagagc atgcccgaga

7801 tcgaaaccga gcaggagtac aaagagatct tcatcacccc ccaccagatc aagcacatta

7861 aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga

7921 ttaacgacac cctgtactcc acccggaagg acgacaaggg caacaccctg atcgtgaaca

7981 atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc 8041 ccgaaaagct gctgatgtac caccacgacc cccagaccta ccagaaactg aagctgatta

8101 tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact

8161 acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg

8221 gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg

8281 tcgtgaagct gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca

8341 agttcgtgac cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata

8401 gcaagtgcta tgaggaagct aagaagctga agaagatcag caaccaggcc gagtttatcg

8461 cctccttcta caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg

8521 tgaacaacga cctgctgaac cggatcgaag tgaacatgat cgacatcacc taccgcgagt

8581 acctggaaaa catgaacgac aagaggcccc ccaggatcat taagacaatc gcctccaaga

8641 cccagagcat taagaagtac agcacagaca ttctgggcaa cctgtatgaa gtgaaatcta

8701 agaagcaccc tcagatcatc aaaaagggca gcggcttcgc caacgagctg ggccctagac

8761 tgatgggaaa gatgcataga ccggtagagc catcacccca gcgctctcca gactcctcta

8821 cgggcatcgg caagaaaggc cagcagcccg caaaaaagag actcaatttt gggcagactg

8881 gcgactcaga gtcagtcccc gaccctcaac caatcggaga accaccagca ggcccctctg

8941 gtctgggatc tggtacaatg gctgcaggcg gtggcgctcc aatggcagac aataacgaag

9001 gcgccgacgg agtgggtagt tcctcaggaa attggcattg cgattccaca tggctgggcg

9061 acagagtcat caccaccagc acccgcacct gggccctgcc cacctacaac aaccacctct

9121 acaagcaaat ctccaacggg acctcgggag gaagcaccaa cgacaacacc tacttcggct

9181 acagcacccc ctgggggtat tttgacttca acagattcca ctgccacttt tcaccacgtg

9241 actggcagcg actcatcaac aacaactggg gattccggcc caagaggctc aacttcaagc

9301 tcttcaacat ccaagtcaag gaggtcacgc agaatgaagg caccaagacc atcgccaata

9361 accttaccag cacgattcag gtctttacgg actcggaata ccagctcccg tacgtgctcg

9421 gctcggcgca ccagggctgc ctgcctccgt tcccggcgga cgtcttcatg attcctcagt

9481 acgggtacct gactctgaac aatggcagtc aggctgtggg ccggtcgtcc ttctactgcc

9541 tggagtactt tccttctcaa atgctgagaa cgggcaacaa ctttgaattc agctacaact

9601 tcgaggacgt gcccttccac agcagctacg cgcacagcca gagcctggac cggctgatga

9661 accctctcat cgaccagtac ttgtactacc tgtcccggac tcaaagcacg ggcggtactg

9721 caggaactca gcagttgcta ttttctcagg ccgggcctaa caacatgtcg gctcaggcca

9781 agaactggct acccggtccc tgctaccggc agcaacgcgt ctccacgaca ctgtcgcaga

9841 acaacaacag caactttgcc tggacgggtg ccaccaagta tcatctgaat ggcagagact

9901 ctctggtgaa tcctggcgtt gccatggcta cccacaagga cgacgaagag cgattttttc

9961 catccagcgg agtcttaatg tttgggaaac agggagctgg aaaagacaac gtggactata

10021 gcagcgtgat gctaaccagc gaggaagaaa taaagaccac caacccagtg gccacagaac 10081 agtacggcgt ggtggccgat aacctgcaac agcaaaacgc cgctcctatt gtaggggccg

10141 tcaatagtca aggagcctta cctggcatgg tgtggcagaa ccgggacgtg tacctgcagg

10201 gtcccatctg ggccaagatt cctcatacgg acggcaactt tcatccctcg ccgctgatgg

10261 gaggctttgg actgaagcat ccgcctcctc agatcctgat taaaaacaca cctgttcccg

10321 cggatcctcc gaccaccttc agccaggcca agctggcttc tttcatcacg cagtacagta

10381 ccggccaggt cagcgtggag atcgagtggg agctgcagaa ggagaacagc aaacgctgga

10441 acccagagat tcagtacact tccaactact acaaatctac aaatgtggac tttgctgtca

10501 atactgaggg tacttattcc gagcctcgcc ccattggcac ccgttacctc acccgtaatc

10561 tgtaattaca tgttaatcaa taaaccggtt aattcgtttc agttgaactt tggtctcctg

10621 tccttcttat cttatcggtt accatagaaa ctggttactt attaactgct tggtgcgctt

10681 cgcgataaaa gacttacgtc atcgggttac ccctagtgat ggagcggccg ctttcagttg

10741 aactttggtc tctgcgtatt tctttcttat ctagtttcca tgctctagag gtcctgtatt

10801 agaggtcacg tgagtgtttt gcgacatttt gcgacaccat gtggtcacgc tgggtattta

10861 agcccgagtg agcacgcagg gtctccattt tgaagcggga ggtttgaacg cgcagccgcc

10921 aagccgaatt ctgcagatat ccatcacact ggcggccgct cgactagagc ggccgccacc

10981 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc

11041 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg

11101 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat

11161 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg

11221 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct

11281 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc

11341 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg

11401 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg

11461 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg

11521 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac

11581 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca

11641 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt

11701 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc

11761 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag

11821 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac

11881 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt

11941 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa

12001 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg

12061 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 12121 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 12181 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc

12241 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat

12301 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc

12361 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc

12421 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag

12481 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg

12541 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg

12601 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag

12661 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt

12721 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga

12781 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc

12841 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc

12901 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc

12961 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc

13021 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca

13081 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat

13141 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt

13201 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt

13261 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg

13321 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc

13381 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca

13441 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga

13501 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa

13561 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc

13621 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt

13681 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt

13741 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg

13801 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac

Staph aureus Cas9 (saCas9)

(SEQ ID NO: 3)

G JPJIVRWEVYISRALWLTTGATMAPKKKRKVGIHGVPAAKRNYILGLDIGITSVGY GIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYN

LLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNEL STKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKA

YHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRS

VKYAYNADLYNALNDLN LVITPJ)ENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEI

LVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSED

IQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKL

VPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREK S

KDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIP

LEDLLN PFNYEVDHIIPRSVSFDNSFN KVLVKQEENSKKGNRTPFQYLSSSDSKISY

ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMN

LLRSYFRVN LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIF

KEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYS

HRVDKKPNP^LINDTLYSTRKDDKGNTLIVN LNGLYDKDNDKLKKLINKSPEKLL

MYHHDPQTYQKLKLIMEQYGDEK PLYKYYEETGNYLTKYSKKDNGPVIKKIKYY

GNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVK LDVIKKENY

YEVNSKCYEEAKKLKKISNQAEFIASFYN DLIKINGELYRVIGVN DLLNRIEVNMI

DITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGSGFA

NELGPRLMGK

pAAVrh74-VPl-3

LOCUS pAAVrh74-VPl-3 10538 bp DNA circular SYN 19-SEP-2016 DEFINITION Knocks out VP2 expression, 5448 A-G

ACCESSION pAAVrh74-VPl-3

REFERENCE 1 (bases 1 to 10538)

FEATURES Location/Qualifiers

misc_feature 84..815

/note="Rep78 5"'

misc_feature 756..815

/note="Rep52 5"'

misc_feature 816..3886

/note="Human Collagen Intron"

misc_feature 3887..5017

/note="Rep52 3"'

misc_feature 3887..5017

/note="Rep78 3"'

misc_feature 4534..4686 /gene="p40"

misc_feature 4741..4742

/note- ' splice donor"

misc_feature 4741..5061

/note="Rep INTRON"

misc_feature 5033..5034

/note- ' splice acceptor"

CDS 5037..7253

/gene="VPl "

misc_feature 5060..5061

/note=" splice acceptor"

CDS 5646.7253

/gene="VP3"

misc_feature complement(7254..7411)

/note="3' UTR"

misc_feature 7428..7507

/note="p5 Promoter"

CDS complement(8893..9753)

/gene="amp"

ORIGIN (SEQ ID NO: 4)

1 cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg 61 ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca 121 gcgaccttga cgagcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga 181 aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc 241 tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg 301 ccccggaggc tcttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg 361 tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc 421 gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg 481 cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca 541 tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg 601 aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc 661 tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg 721 cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg

781 acaaggggat tacctcggag aagcagtgga tccaggtgag taattgacaa agccaaacac 841 caccatttgc cgagcacttt agagtttaca ggtttgtttc tcttgaccct caaaacaaac

901 ctgtgaggca tagggagtat tgctatccct taagaattca cccccagtgt gcccatcaaa

961 acctcccagg ctgagtctgc acagttgaag gaggaaggat aggaatggga gggtcgatgg

1021 gtgaaagcat gattctctta accagtccag attatcaggt aatcccttca acaaccacca

1081 cccactccct gggcaatcca gctggagttt acagacagac ttagctggct atagcaccac

1141 cgtgctactc tctgttcttc ctggttgctc aaatgcccta gaaaagtgga acaggtgagc

1201 atcaactcac agggctctat gctggctgct gctgcgaggg atgttatgct atagtaccag

1261 gggccaccat tccataggca cttcctgtgt ttaataccct atatgcttta cttcatctca

1321 tcttcctcca tatcctgaga ggtggttcta ttcttctccc cattttacgg atg aaaaaac

1381 cgagacacag aaaggtgaaa tagcttaaga taaatggtgc cttgcagcct tagactctgg

1441 tggcctctag ttaatgtggg aaattaaggg tgaggggatt ggcagctgat ggagggtgca

1501 gggtgccaga cagaggcgtt tagctctgat cccttagcaa tagagagtcc ttgtaggcac

1561 ttggtcaggc gagtgatgcg atgaaagctg tgtttaagaa agattatgct ttctgctgat

1621 ttcatacccc caacacccaa gctctgaggc ccctcctcac aggtccttgc agggctggcc

1681 aaaataaagc agcttcactc cgttgtgctg ctttccagct aatgtgtctg tttggcagaa

1741 gtttccctca aaggcagatc agtgaaataa gcagaagcct cgacccccct ttgtcagcca

1801 gagctgctga agtgccttgc cccagggtca ctttgtgtga ggggattaga gagcactggg

1861 gctgccaaga aacactgccg tttctacaga ttagcaggac gctggcttgt ggccttctag

1921 cgaggctcag agctgcggtg gccctagtct gcatgggcta aagacaagct ccatctcctg

1981 tccttgttcc ctccttcctg ggcacagccg ccctgcttct tggttctctc tgttggttcc

2041 tgtccgcacg gtagttaggc tggcagcgtg tgtaggattt ggcttagaag attgacaaca

2101 ttgcctttga gcccttcttt gctactcctc cctctcccct cccatcagac tcctctctgg

2161 agtctgctct gcgaggcctc tgctctgtgg tatcccagca gccttctcag ccttgacttc

2221 cagaaggggg ctgtgcagtg tccggggtgt gcaggcccca gacacggggt aggctcatgg

2281 agatccaagt gctgatctag tgtcaaggct ggcctggaga ctgggctggg ttggtgtctg

2341 cctgctgtgg tcatgtgccc tcccttgggc ctgtatcctc tctccagact tgctgcaggg

2401 agaggtggca gatgtcagcc tagttctggc ctctcagagc agcatggcag ctccctttca

2461 ctcaggccca ggctgggccc tcctgctggc tgacccctgg ggagagggtg ctccagagct

2521 ccccaaggaa cagcttcccg aagcagccag gccagcccag aggggctgtg gccaatcctg

2581 aagctttatg ttcctgctga cattttttct aagttttctc ttgctttcct cttaaatgcc

2641 aatctggaga gtctccgtta ggagaaatgg accccagcca ggaagaagag ttgagttgta

2701 tttaaaacac gagctccccc taaagcatcc ttctttagct tctaaggaga ggcagagact

2761 gacaggcagg actcagcagg aaaaggtacc cccctgacct gctcagtcag gccctaggcc

2821 cagctccacc cagcctgtgg cccccagagt ttcggtaaag agttccctgg gccttaagga 2881 accttgagag agcatttgag gggtgccacc acaaacttgg cagaaaaaac cctccccctc

2941 caagtccagt cctagagaag gagctggcaa ccttgccttg ctttgtaagc aaaagcctct

3001 tagggcttga gctcagatgt agtgtttgag ctgtggctgg tgccctgccc catcagggag

3061 ccaatggtag acatcctatg ggcatctttg ttttccgtaa gagcaggctg tctggggatg

3121 ggccagagga agaggcgacc tggagtcaac caagaggagg ccttaaccaa gccttaacca

3181 cagaggttaa ccaagccttg aaagcgcttc cccctgagca ggcaggaagc actgagtcca

3241 catggttgcc tcgctgtttc atttccttac actcaattct ctcagtcttt aaatgatcac

3301 ttggccttga agttacggat atttggggtc tgaactgaag ttgaagaaaa gaggaaatga

3361 tttaagcttt gtttaagatt aggggccagg tgcggtggct cacgcctgta atcccagcac

3421 cttgggagcc tgaggcgggt ggatcacctg aggtcaggag ttccagacca gcctggccaa

3481 catagcaaaa cccagtctct actaaaaata acaataaaaa aattagccag gtgtggtgac

3541 acatgcctgt aatcccagtt actcaggagg ctgaggcaga attgcttgaa cttgagaggt

3601 ggaggttgta gtgagccaag accgcaccac tgcactccag cctggcgaca gagccagact

3661 ccgtctcaaa aacaacaaca aaaaagatta gaagaagccc attactgcct tctggccacc

3721 cactcgcaca gacaccaaaa ctgcagccca cacctcgcca tcctcgtgct ctgccctggg

3781 acaccccagg cacagtgtgt ccttcgtttt ctgtaagggt gggctgggag cagggacgga

3841 cagggcctgt gggcacctct catggtcact tccttcttgc tcacaggagg accaggcctc

3901 atacatctcc ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa

3961 tgcgggaaag attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc

4021 cgtggaggac atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc

4081 ccaatatgcg gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac

4141 catctggctg tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca

4201 cactgtgccc ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg

4261 tgtcgacaag atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc

4321 ggccaaagcc attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc

4381 ccagatagac ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga

4441 cgggaactca acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga

4501 actcacccgc cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt

4561 tttccggtgg gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg

4621 tggagccaag aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg

4681 cgagtcagtt gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag

4741 gtaccaaaac aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca

4801 atgcgagaga atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt

4861 agagtgcttt cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa 4921 actgtgctac attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct

4981 ggtcaatgtg gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtatgg

5041 ctgccgatgg ttatcttcca gattggctcg aggacaacct ctctgagggc attcgcgagt

5101 ggtgggacct gaaacctgga gccccgaaac ccaaagccaa ccagcaaaag caggacaacg

5161 gccggggtct ggtgcttcct ggctacaagt acctcggacc cttcaacgga ctcgacaagg

5221 gggagcccgt caacgcggcg gacgcagcgg ccctcgagca cgacaaggcc tacgaccagc

5281 agctccaagc gggtgacaat ccgtacctgc ggtataatca cgccgacgcc gagtttcagg

5341 agcgtctgca agaagatacg tcttttgggg gcaacctcgg gcgcgcagtc ttccaggcca

5401 aaaagcgggt tctcgaacct ctgggcctgg ttgaatcgcc ggttaaggcg gctcctggaa

5461 agaagagacc ggtagagcca tcaccccagc gctctccaga ctcctctacg ggcatcggca

5521 agaaaggcca gcagcccgca aaaaagagac tcaattttgg gcagactggc gactcagagt

5581 cagtccccga ccctcaacca atcggagaac caccagcagg cccctctggt ctgggatctg

5641 gtacaatggc tgcaggcggt ggcgctccaa tggcagacaa taacgaaggc gccgacggag

5701 tgggtagttc ctcaggaaat tggcattgcg attccacatg gctgggcgac agagtcatca

5761 ccaccagcac ccgcacctgg gccctgccca cctacaacaa ccacctctac aagcaaatct

5821 ccaacgggac ctcgggagga agcaccaacg acaacaccta cttcggctac agcaccccct

5881 gggggtattt tgacttcaac agattccact gccacttttc accacgtgac tggcagcgac

5941 tcatcaacaa caactgggga ttccggccca agaggctcaa cttcaagctc ttcaacatcc

6001 aagtcaagga ggtcacgcag aatgaaggca ccaagaccat cgccaataac cttaccagca

6061 cgattcaggt ctttacggac tcggaatacc agctcccgta cgtgctcggc tcggcgcacc

6121 agggctgcct gcctccgttc ccggcggacg tcttcatgat tcctcagtac gggtacctga

6181 ctctgaacaa tggcagtcag gctgtgggcc ggtcgtcctt ctactgcctg gagtactttc

6241 cttctcaaat gctgagaacg ggcaacaact ttgaattcag ctacaacttc gaggacgtgc

6301 ccttccacag cagctacgcg cacagccaga gcctggaccg gctgatgaac cctctcatcg

6361 accagtactt gtactacctg tcccggactc aaagcacggg cggtactgca ggaactcagc

6421 agttgctatt ttctcaggcc gggcctaaca acatgtcggc tcaggccaag aactggctac

6481 ccggtccctg ctaccggcag caacgcgtct ccacgacact gtcgcagaac aacaacagca

6541 actttgcctg gacgggtgcc accaagtatc atctgaatgg cagagactct ctggtgaatc

6601 ctggcgttgc catggctacc cacaaggacg acgaagagcg attttttcca tccagcggag

6661 tcttaatgtt tgggaaacag ggagctggaa aagacaacgt ggactatagc agcgtgatgc

6721 taaccagcga ggaagaaata aagaccacca acccagtggc cacagaacag tacggcgtgg

6781 tggccgataa cctgcaacag caaaacgccg ctcctattgt aggggccgtc aatagtcaag

6841 gagccttacc tggcatggtg tggcagaacc gggacgtgta cctgcagggt cccatctggg

6901 ccaagattcc tcatacggac ggcaactttc atccctcgcc gctgatggga ggctttggac 6961 tgaagcatcc gcctcctcag atcctgatta aaaacacacc tgttcccgcg gatcctccga

7021 ccaccttcag ccaggccaag ctggcttctt tcatcacgca gtacagtacc ggccaggtca

7081 gcgtggagat cgagtgggag ctgcagaagg agaacagcaa acgctggaac ccagagattc

7141 agtacacttc caactactac aaatctacaa atgtggactt tgctgtcaat actgagggta

7201 cttattccga gcctcgcccc attggcaccc gttacctcac ccgtaatctg taattacatg

7261 ttaatcaata aaccggttaa ttcgtttcag ttgaactttg gtctcctgtc cttcttatct

7321 tatcggttac catagaaact ggttacttat taactgcttg gtgcgcttcg cgataaaaga

7381 cttacgtcat cgggttaccc ctagtgatgg agcggccgct ttcagttgaa ctttggtctc

7441 tgcgtatttc tttcttatct agtttccatg ctctagaggt cctgtattag aggtcacgtg

7501 agtgttttgc gacattttgc gacaccatgt ggtcacgctg ggtatttaag cccgagtgag

7561 cacgcagggt ctccattttg aagcgggagg tttgaacgcg cagccgccaa gccgaattct

7621 gcagatatcc atcacactgg cggccgctcg actagagcgg ccgccaccgc ggtggagctc

7681 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct

7741 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat

7801 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc

7861 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg

7921 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct

7981 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt

8041 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc

8101 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga

8161 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata

8221 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac

8281 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg

8341 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc

8401 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag

8461 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt

8521 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt

8581 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg

8641 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac

8701 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca

8761 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac

8821 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac

8881 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt

8941 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 9001 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt

9061 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc

9121 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa

9181 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg

9241 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt

9301 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc

9361 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt

9421 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg

9481 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac

9541 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc

9601 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt

9661 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg

9721 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag

9781 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa

9841 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat

9901 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc

9961 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt

10021 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa

10081 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg

10141 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga

10201 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 10261 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat 10321 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 10381 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga

10441 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag

10501 cgcgcgtaat acgactcact atagggcgaa ttgggtac

pAAVrh74-Cas9-VP2

LOCUS pAAVrh74-Cas9-VP 13859 bp DNA circular SYN 09-MAR-2017 REFERENCE 1 (bases 1 to 13859)

FEATURES Location/Qualifiers

misc_feature 84..815

/note="Rep68 5"'

misc_feature 84..815

/note="Rep78 5"' misc_feature 756..815

/note="Rep40 5"'

misc_feature 756..815

/note="Rep52 5"'

misc feature 816..3886

/note="Human Collagen Intron" misc_feature 3887..5017

/note="Rep52 3"'

misc_feature 3887..5017

/note="Rep78 3"'

misc_feature 4534..4686

/gene="p40 pro"

misc_feature 4741..4742

/note- ' splice donor"

misc_feature 4741..5061

/note="Rep INTRON" misc_feature 5033..5034

/note="splice acceptor" misc_feature 5060..5061

/note="splice acceptor" misc_feature 5084..5086

/note="Rep 68/40 stop"

CDS 5532..8781

/gene="'saCas9"

misc_feature 8739..8780

/product- ' OLLAS tag epitope tag" CDS 8786..10574

/gene="'rh74 cap"

/codon_start=3

/translation="DR"

misc_feature complement(l 0575..10732)

/note="3' UTR"

misc_feature 10749..10828

/note="p5 Promoter" CDS complement(12214..13074)

/gene="amp"

ORIGIN (SEQ ID NO: 5)

1 cgggcccccc ctcgaggtcg acggtatcgg gggagctcgc agggtctcca ttttgaagcg

61 ggaggtttga acgcgcagcc gccatgccgg ggttttacga gattgtgatt aaggtcccca

121 gcgaccttga cgagcatctg cccggcattt ctgacagctt tgtgaactgg gtggccgaga

181 aggaatggga gttgccgcca gattctgaca tggatctgaa tctgattgag caggcacccc

241 tgaccgtggc cgagaagctg cagcgcgact ttctgacgga atggcgccgt gtgagtaagg

301 ccccggaggc tcttttcttt gtgcaatttg agaagggaga gagctacttc cacatgcacg

361 tgctcgtgga aaccaccggg gtgaaatcca tggttttggg acgtttcctg agtcagattc

421 gcgaaaaact gattcagaga atttaccgcg ggatcgagcc gactttgcca aactggttcg

481 cggtcacaaa gaccagaaat ggcgccggag gcgggaacaa ggtggtggat gagtgctaca

541 tccccaatta cttgctcccc aaaacccagc ctgagctcca gtgggcgtgg actaatatgg

601 aacagtattt aagcgcctgt ttgaatctca cggagcgtaa acggttggtg gcgcagcatc

661 tgacgcacgt gtcgcagacg caggagcaga acaaagagaa tcagaatccc aattctgatg

721 cgccggtgat cagatcaaaa acttcagcca ggtacatgga gctggtcggg tggctcgtgg

781 acaaggggat tacctcggag aagcagtgga tccaggtgag taattgacaa agccaaacac

841 caccatttgc cgagcacttt agagtttaca ggtttgtttc tcttgaccct caaaacaaac

901 ctgtgaggca tagggagtat tgctatccct taagaattca cccccagtgt gcccatcaaa

961 acctcccagg ctgagtctgc acagttgaag gaggaaggat aggaatggga gggtcgatgg

1021 gtgaaagcat gattctctta accagtccag attatcaggt aatcccttca acaaccacca

1081 cccactccct gggcaatcca gctggagttt acagacagac ttagctggct atagcaccac

1141 cgtgctactc tctgttcttc ctggttgctc aaatgcccta gaaaagtgga acaggtgagc

1201 atcaactcac agggctctat gctggctgct gctgcgaggg atgttatgct atagtaccag

1261 gggccaccat tccataggca cttcctgtgt ttaataccct atatgcttta cttcatctca

1321 tcttcctcca tatcctgaga ggtggttcta ttcttctccc cattttacgg atg aaaaaac

1381 cgagacacag aaaggtgaaa tagcttaaga taaatggtgc cttgcagcct tagactctgg

1441 tggcctctag ttaatgtggg aaattaaggg tgaggggatt ggcagctgat ggagggtgca

1501 gggtgccaga cagaggcgtt tagctctgat cccttagcaa tagagagtcc ttgtaggcac

1561 ttggtcaggc gagtgatgcg atgaaagctg tgtttaagaa agattatgct ttctgctgat

1621 ttcatacccc caacacccaa gctctgaggc ccctcctcac aggtccttgc agggctggcc

1681 aaaataaagc agcttcactc cgttgtgctg ctttccagct aatgtgtctg tttggcagaa

1741 gtttccctca aaggcagatc agtgaaataa gcagaagcct cgacccccct ttgtcagcca

1801 gagctgctga agtgccttgc cccagggtca ctttgtgtga ggggattaga gagcactggg 1861 gctgccaaga aacactgccg tttctacaga ttagcaggac gctggcttgt ggccttctag

1921 cgaggctcag agctgcggtg gccctagtct gcatgggcta aagacaagct ccatctcctg

1981 tccttgttcc ctccttcctg ggcacagccg ccctgcttct tggttctctc tgttggttcc

2041 tgtccgcacg gtagttaggc tggcagcgtg tgtaggattt ggcttagaag attgacaaca

2101 ttgcctttga gcccttcttt gctactcctc cctctcccct cccatcagac tcctctctgg

2161 agtctgctct gcgaggcctc tgctctgtgg tatcccagca gccttctcag ccttgacttc

2221 cagaaggggg ctgtgcagtg tccggggtgt gcaggcccca gacacggggt aggctcatgg

2281 agatccaagt gctgatctag tgtcaaggct ggcctggaga ctgggctggg ttggtgtctg

2341 cctgctgtgg tcatgtgccc tcccttgggc ctgtatcctc tctccagact tgctgcaggg

2401 agaggtggca gatgtcagcc tagttctggc ctctcagagc agcatggcag ctccctttca

2461 ctcaggccca ggctgggccc tcctgctggc tgacccctgg ggagagggtg ctccagagct

2521 ccccaaggaa cagcttcccg aagcagccag gccagcccag aggggctgtg gccaatcctg

2581 aagctttatg ttcctgctga cattttttct aagttttctc ttgctttcct cttaaatgcc

2641 aatctggaga gtctccgtta ggagaaatgg accccagcca ggaagaagag ttgagttgta

2701 tttaaaacac gagctccccc taaagcatcc ttctttagct tctaaggaga ggcagagact

2761 gacaggcagg actcagcagg aaaaggtacc cccctgacct gctcagtcag gccctaggcc

2821 cagctccacc cagcctgtgg cccccagagt ttcggtaaag agttccctgg gccttaagga

2881 accttgagag agcatttgag gggtgccacc acaaacttgg cagaaaaaac cctccccctc

2941 caagtccagt cctagagaag gagctggcaa ccttgccttg ctttgtaagc aaaagcctct

3001 tagggcttga gctcagatgt agtgtttgag ctgtggctgg tgccctgccc catcagggag

3061 ccaatggtag acatcctatg ggcatctttg ttttccgtaa gagcaggctg tctggggatg

3121 ggccagagga agaggcgacc tggagtcaac caagaggagg ccttaaccaa gccttaacca

3181 cagaggttaa ccaagccttg aaagcgcttc cccctgagca ggcaggaagc actgagtcca

3241 catggttgcc tcgctgtttc atttccttac actcaattct ctcagtcttt aaatgatcac

3301 ttggccttga agttacggat atttggggtc tgaactgaag ttgaagaaaa gaggaaatga

3361 tttaagcttt gtttaagatt aggggccagg tgcggtggct cacgcctgta atcccagcac

3421 cttgggagcc tgaggcgggt ggatcacctg aggtcaggag ttccagacca gcctggccaa

3481 catagcaaaa cccagtctct actaaaaata acaataaaaa aattagccag gtgtggtgac

3541 acatgcctgt aatcccagtt actcaggagg ctgaggcaga attgcttgaa cttgagaggt

3601 ggaggttgta gtgagccaag accgcaccac tgcactccag cctggcgaca gagccagact

3661 ccgtctcaaa aacaacaaca aaaaagatta gaagaagccc attactgcct tctggccacc

3721 cactcgcaca gacaccaaaa ctgcagccca cacctcgcca tcctcgtgct ctgccctggg

3781 acaccccagg cacagtgtgt ccttcgtttt ctgtaagggt gggctgggag cagggacgga

3841 cagggcctgt gggcacctct catggtcact tccttcttgc tcacaggagg accaggcctc 3901 atacatctcc ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa

3961 tgcgggaaag attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc

4021 cgtggaggac atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc

4081 ccaatatgcg gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac

4141 catctggctg tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca

4201 cactgtgccc ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg

4261 tgtcgacaag atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc

4321 ggccaaagcc attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc

4381 ccagatagac ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga

4441 cgggaactca acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga

4501 actcacccgc cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt

4561 tttccggtgg gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg

4621 tggagccaag aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg

4681 cgagtcagtt gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag

4741 gtaccaaaac aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca

4801 atgcgagaga atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt

4861 agagtgcttt cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa

4921 actgtgctac attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct

4981 ggtcaatgtg gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtctgg

5041 ctgccgatgg ttatcttcca gattggctcg aggacaacct ctctgagggc attcgcgagt

5101 ggtgggacct gaaacctgga gccccgaaac ccaaagccaa ccagcaaaag caggacaacg

5161 gccggggtct ggtgcttcct ggctacaagt acctcggacc cttcaacgga ctcgacaagg

5221 gggagcccgt caacgcggcg gacgcagcgg ccctcgagca cgacaaggcc tacgaccagc

5281 agctccaagc gggtgacaat ccgtacctgc ggtataatca cgccgacgcc gagtttcagg

5341 agcgtctgca agaagatacg tcttttgggg gcaacctcgg gcgcgcagtc ttccaggcca

5401 aaaagcgggt tctcgaacct ctgggcctgg ttgaatcgcc ggttaagatg gctagcggaa

5461 ctagcggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta

5521 ccggtgccac catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag

5581 ccaagcggaa ctacatcctg ggcctggaca tcggcatcac cagcgtgggc tacggcatca

5641 tcgactacga gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg

5701 tggaaaacaa cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc

5761 ggcatagaat ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca

5821 gcgagctgag cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga

5881 gcgaggaaga gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg 5941 tgaacgaggt ggaagaggac accggcaacg agctgtccac caaagagcag atcagccgga

6001 acagcaaggc cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag

6061 acggcgaagt gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca

6121 aacagctgct gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct

6181 acatcgacct gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct

6241 tcggctggaa ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc

6301 ccgaggaact gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg

6361 acctgaacaa tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt

6421 tccagatcat cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca

6481 aagaaatcct cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc

6541 ccgagttcac caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga

6601 ttattgagaa cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca

6661 gcgaggacat ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg

6721 agcagatctc taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca

6781 acctgatcct ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc

6841 tgaagctggt gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg

6901 tggacgactt catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga

6961 tcaacgccat catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg

7021 agaagaactc caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga

7081 ccaacgagcg gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga

7141 tcgagaagat caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca

7201 tccctctgga agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca

7261 gaagcgtgtc cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaaaaca

7321 gcaagaaggg caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct

7381 acgaaacctt caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga

7441 ccaagaaaga gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact

7501 tcatcaaccg gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc

7561 ggagctactt cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca

7621 ccagctttct gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc

7681 acgccgagga cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac

7741 tggacaaggc caaaaaagtg atggaaaacc agatgttcga ggaaaagcag gccgagagca

7801 tgcccgagat cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca

7861 agcacattaa ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata

7921 gagagctgat taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga 7981 tcgtgaacaa tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca

8041 acaagagccc cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga

8101 agctgattat ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa

8161 ccgggaacta cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta

8221 agtattacgg caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca

8281 gaaacaaggt cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg

8341 gcgtgtacaa gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg

8401 aagtgaatag caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg

8461 agtttatcgc ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag

8521 tgatcggcgt gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct

8581 accgcgagta cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg

8641 cctccaagac ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag

8701 tgaaatctaa gaagcaccct cagatcatca aaaagggcag cggcttcgcc aacgagctgg

8761 gccctagact gatgggaaag actagtagac cggtagagcc atcaccccag cgctctccag

8821 actcctctac gggcatcggc aagaaaggcc agcagcccgc aaaaaagaga ctcaattttg

8881 ggcagactgg cgactcagag tcagtccccg accctcaacc aatcggagaa ccaccagcag

8941 gcccctctgg tctgggatct ggtacactgg ctgcaggcgg tggcgctcca ctggcagaca

9001 ataacgaagg cgccgacgga gtgggtagtt cctcaggaaa ttggcattgc gattccacat

9061 ggctgggcga cagagtcatc accaccagca cccgcacctg ggccctgccc acctacaaca

9121 accacctcta caagcaaatc tccaacggga cctcgggagg aagcaccaac gacaacacct

9181 acttcggcta cagcaccccc tgggggtatt ttgacttcaa cagattccac tgccactttt

9241 caccacgtga ctggcagcga ctcatcaaca acaactgggg attccggccc aagaggctca

9301 acttcaagct cttcaacatc caagtcaagg aggtcacgca gaatgaaggc accaagacca

9361 tcgccaataa ccttaccagc acgattcagg tctttacgga ctcggaatac cagctcccgt

9421 acgtgctcgg ctcggcgcac cagggctgcc tgcctccgtt cccggcggac gtcttcatga

9481 ttcctcagta cgggtacctg actctgaaca atggcagtca ggctgtgggc cggtcgtcct

9541 tctactgcct ggagtacttt ccttctcaaa tgctgagaac gggcaacaac tttgaattca

9601 gctacaactt cgaggacgtg cccttccaca gcagctacgc gcacagccag agcctggacc

9661 ggctgatgaa ccctctcatc gaccagtact tgtactacct gtcccggact caaagcacgg

9721 gcggtactgc aggaactcag cagttgctat tttctcaggc cgggcctaac aacatgtcgg

9781 ctcaggccaa gaactggcta cccggtccct gctaccggca gcaacgcgtc tccacgacac

9841 tgtcgcagaa caacaacagc aactttgcct ggacgggtgc caccaagtat catctgaatg

9901 gcagagactc tctggtgaat cctggcgttg ccatggctac ccacaaggac gacgaagagc

9961 gattttttcc atccagcgga gtcttaatgt ttgggaaaca gggagctgga aaagacaacg 10021 tggactatag cagcgtgatg ctaaccagcg aggaagaaat aaagaccacc aacccagtgg

10081 ccacagaaca gtacggcgtg gtggccgata acctgcaaca gcaaaacgcc gctcctattg

10141 taggggccgt caatagtcaa ggagccttac ctggcatggt gtggcagaac cgggacgtgt

10201 acctgcaggg tcccatctgg gccaagattc ctcatacgga cggcaacttt catccctcgc

10261 cgctgatggg aggctttgga ctgaagcatc cgcctcctca gatcctgatt aaaaacacac

10321 ctgttcccgc ggatcctccg accaccttca gccaggccaa gctggcttct ttcatcacgc

10381 agtacagtac cggccaggtc agcgtggaga tcgagtggga gctgcagaag gagaacagca

10441 aacgctggaa cccagagatt cagtacactt ccaactacta caaatctaca aatgtggact

10501 ttgctgtcaa tactgagggt acttattccg agcctcgccc cattggcacc cgttacctca

10561 cccgtaatct gtaattacat gttaatcaat aaaccggtta attcgtttca gttgaacttt

10621 ggtctcctgt ccttcttatc ttatcggtta ccatagaaac tggttactta ttaactgctt

10681 ggtgcgcttc gcgataaaag acttacgtca tcgggttacc cctagtgatg gagcggccgc

10741 tttcagttga actttggtct ctgcgtattt ctttcttatc tagtttccat gctctagagg

10801 tcctgtatta gaggtcacgt gagtgttttg cgacattttg cgacaccatg tggtcacgct

10861 gggtatttaa gcccgagtga gcacgcaggg tctccatttt gaagcgggag gtttgaacgc

10921 gcagccgcca agccgaattc tgcagatatc catcacactg gcggccgctc gactagagcg

10981 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt

11041 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca

11101 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact

11161 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct

11221 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc

11281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca

11341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg

11401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca

11461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa

11521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc

11581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc

11641 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct

11701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg

11761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag

11821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta

11881 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg

11941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt

12001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 12061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 12121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 12181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 12241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 12301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 12361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 12421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 12481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 12541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 12601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 12661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 12721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 12781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 12841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 12901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 12961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 13021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 13081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 13141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 13201 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc

13261 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 13321 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 13381 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 13441 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 13501 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 13561 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 13621 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg 13681 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 13741 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 13801 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtac pHELP

LOCUS pHELP 11635 bp DNA circular SYN 19- JUL-2016

REFERENCE 1 (bases 1 to 11635)

FEATURES Location/Qualifiers misc_feature complement(258..1841)

/note="Ad5 E2A DBP"

misc_feature 839..903

/note="E2A Primer / probe Region"

misc_feature 5647..8267

/note="Ad5 E4 Gene"

misc_feature complement(8546..8662)

/note="52K Partial"

misc_feature 8661..9121

/note="VA RNA Region"

CDS complement(10182..11042)

/gene="amp"

ORIGIN (SEQ ID NO: 6)

1 ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga

61 acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat

121 taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctaggagaca

181 ctttcaataa aggcaaatgt ttttatttgt acactctcgg gtgattattt accccccacc

241 cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact

301 ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc

361 cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc

421 aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg

481 cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc

541 acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga

601 gtcaactttg gtagctgcct tcccaaaaag ggtgcatgcc caggctttga gttgcactcg

661 caccgtagtg gcatcagaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc

721 atgaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg

781 ccgcaagact tgccggaaaa ctgattggcc ggacaggccg cgtcatgcac gcagcacctt

841 gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc

901 ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc

961 acgtgctcct tatttatcat aatgctcccg tgtagacact taagctcgcc ttcgatctca

1021 gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt ggtgcttgta ggttacctct

1081 gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg

1141 ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttta gccaggtctt gcatacggcc

1201 gccagagctt ccacttggtc aggcagtagc ttgaagtttg cctttagatc gttatccacg 1261 tggtacttgt ccatcaacgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc

1321 ggcaggctca gcgggtttat caccgtgctt tcactttccg cttcactgga ctcttccttt

1381 tcctcttgcg tccgcatacc ccgcgccact gggtcgtctt cattcagccg ccgcaccgtg

1441 cgcttacctc ccttgccgtg cttgattagc accggtgggt tgctgaaacc caccatttgt

1501 agcgccacat cttctctttc ttcctcgctg tccacgatca cctctgggga tggcgggcgc

1561 tcgggcttgg gagaggggcg cttctttttc tttttggacg caatggccaa atccgccgtc

1621 gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg catcttgtga cgagtcttct

1681 tcgtcctcgg actcgagacg ccgcctcagc cgcttttttg ggggcgcgcg gggaggcggc

1741 ggcgacggcg acggggacga cacgtcctcc atggttggtg gacgtcgcgc cgcaccgcgt

1801 ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat

1861 aggcagaaaa agatcatgga gtcagtcgag aaggaggaca gcctaaccgc cccctttgag

1921 ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca

1981 cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac

2041 gacgaggatc gctcagtacc aacagaggat aaaaagcaag accaggacga cgcagaggca

2101 aacgaggaac aagtcgggcg gggggaccaa aggcatggcg actacctaga tgtgggagac

2161 gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag

2221 cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctgttc

2281 tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc

2341 aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa

2401 aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc

2461 ttgcggcagg gcgctgtcat acctgatatc gcctcgctcg acgaagtgcc aaaaatcttt

2521 gagggtcttg gacgcgacga gaaacgcgcg gcaaacgctc tgcaacaaga aaacagcgaa

2581 aatgaaagtc actgtggagt gctggtggaa cttgagggtg acaacgcgcg cctagccgtg

2641 ctgaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag

2701 gttatgagca cagtcatgag cgagctgatc gtgcgccgtg cacgacccct ggagagggat

2761 gcaaacttgc aagaacaaac cgaggagggc ctacccgcag ttggcgatga gcagctggcg

2821 cgctggcttg agacgcgcga gcctgccgac ttggaggagc gacgcaagct aatgatggcc

2881 gcagtgcttg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg

2941 cagcgcaagc tagaggaaac gttgcactac acctttcgcc agggctacgt gcgccaggcc

3001 tgcaaaattt ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa

3061 aaccgcctcg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac

3121 gtccgcgact gcgtttactt atttctgtgc tacacctggc aaacggccat gggcgtgtgg

3181 cagcaatgcc tggaggagcg caacctaaag gagctgcaga agctgctaaa gcaaaacttg

3241 aaggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacattatc 3301 ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc

3361 atgttgcaaa actttaggaa ctttatccta gagcgttcag gaattctgcc cgccacctgc

3421 tgtgcgcttc ctagcgactt tgtgcccatt aagtaccgtg aatgccctcc gccgctttgg

3481 ggtcactgct accttctgca gctagccaac taccttgcct accactccga catcatggaa

3541 gacgtgagcg gtgacggcct actggagtgt cactgtcgct gcaacctatg caccccgcac

3601 cgctccctgg tctgcaattc gcaactgctt agcgaaagtc aaattatcgg tacctttgag

3661 ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg

3721 ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt

3781 aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc

3841 cagggccaca tccttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta

3901 cgaaagggac ggggggttta cctggacccc cagtccggcg aggagctcaa cccaatcccc

3961 ccgccgccgc agccctatca gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa

4021 gaagctgcag ctgccgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc

4081 agaggaggtt ttggacgagg aggaggagat gatggaagac tgggacagcc tagacgaagc

4141 ttccgaggcc gaagaggtgt cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc

4201 ggcgccccag aaattggcaa ccgttcccag catcgctaca acctccgctc ctcaggcgcc

4261 gccggcactg cctgttcgcc gacccaaccg tagatgggac accactggaa ccagggccgg

4321 taagtctaag cagccgccgc cgttagccca agagcaacaa cagcgccaag gctaccgctc

4381 gtggcgcggg cacaagaacg ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc

4441 cttcgcccgc cgctttcttc tctaccatca cggcgtggcc ttcccccgta acatcctgca

4501 ttactaccgt catctctaca gcccctactg caccggcggc agcggcagcg gcagcaacag

4561 cagcggtcac acagaagcaa aggcgaccgg atagcaagac tctgacaaag cccaagaaat

4621 ccacagcggc ggcagcagca ggaggaggag cgctgcgtct ggcgcccaac gaacccgtat

4681 cgacccgcga gcttagaaat aggatttttc ccactctgta tgctatattt caacaaagca

4741 ggggccaaga acaagagctg aaaataaaaa acaggtctct gcgctccctc acccgcagct

4801 gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct ggaagacgcg gaggctctct

4861 tcagcaaata ctgcgcgctg actcttaagg actagtttcg cgccctttct caaatttaag

4921 cgcgaaaact acgtcatctc cagcggccac acccggcgcc agcacctgtc gtcagcgcca

4981 ttatgagcaa ggaaattccc acgccctaca tgtggagtta ccagccacaa atgggacttg

5041 cggctggagc tgcccaagac tactcaaccc gaataaacta catgagcgcg ggaccccaca

5101 tgatatcccg ggtcaacgga atccgcgccc accgaaaccg aattctcctc gaacaggcgg

5161 ctattaccac cacacctcgt aataacctta atccccgtag ttggcccgct gccctggtgt

5221 accaggaaag tcccgctccc accactgtgg tacttcccag agacgcccag gccgaagttc

5281 agatgactaa ctcaggggcg cagcttgcgg gcggctttcg tcacagggtg cggtcgcccg 5341 ggcgttttag ggcggagtaa cttgcatgta ttgggaattg tagttttttt aaaatgggaa

5401 gtgacgtatc gtgggaaaac ggaagtgaag atttgaggaa gttgtgggtt ttttggcttt

5461 cgtttctggg cgtaggttcg cgtgcggttt tctgggtgtt ttttgtggac tttaaccgtt

5521 acgtcatttt ttagtcctat atatactcgc tctgtacttg gcccttttta cactgtgact

5581 gattgagctg gtgccgtgtc gagtggtgtt ttttaatagg tttttttact ggtaaggctg

5641 actgttatgg ctgccgctgt ggaagcgctg tatgttgttc tggagcggga gggtgctatt

5701 ttgcctaggc aggagggttt ttcaggtgtt tatgtgtttt tctctcctat taattttgtt

5761 atacctccta tgggggctgt aatgttgtct ctacgcctgc gggtatgtat tcccccgggc

5821 tatttcggtc gctttttagc actgaccgat gttaaccaac ctgatgtgtt taccgagtct

5881 tacattatga ctccggacat gaccgaggaa ctgtcggtgg tgctttttaa tcacggtgac

5941 cagttttttt acggtcacgc cggcatggcc gtagtccgtc ttatgcttat aagggttgtt

6001 tttcctgttg taagacaggc ttctaatgtt taaatgtttt tttttttgtt attttatttt

6061 gtgtttaatg caggaacccg cagacatgtt tgagagaaaa atggtgtctt tttctgtggt

6121 ggttccggaa cttacctgcc tttatctgca tgagcatgac tacgatgtgc ttgctttttt

6181 gcgcgaggct ttgcctgatt ttttgagcag caccttgcat tttatatcgc cgcccatgca

6241 acaagcttac ataggggcta cgctggttag catagctccg agtatgcgtg tcataatcag

6301 tgtgggttct tttgtcatgg ttcctggcgg ggaagtggcc gcgctggtcc gtgcagacct

6361 gcacgattat gttcagctgg ccctgcgaag ggacctacgg gatcgcggta tttttgttaa

6421 tgttccgctt ttgaatctta tacaggtctg tgaggaacct gaatttttgc aatcatgatt

6481 cgctgcttga ggctgaaggt ggagggcgct ctggagcaga tttttacaat ggccggactt

6541 aatattcggg atttgcttag agacatattg ataaggtggc gagatgaaaa ttatttgggc

6601 atggttgaag gtgctggaat gtttatagag gagattcacc ctgaagggtt tagcctttac

6661 gtccacttgg acgtgagggc agtttgcctt ttggaagcca ttgtgcaaca tcttacaaat

6721 gccattatct gttctttggc tgtagagttt gaccacgcca ccggagggga gcgcgttcac

6781 ttaatagatc ttcattttga ggttttggat aatcttttgg aataaaaaaa aaaaaacatg

6841 gttcttccag ctcttcccgc tcctcccgtg tgtgactcgc agaacgaatg tgtaggttgg

6901 ctgggtgtgg cttattctgc ggtggtggat gttatcaggg cagcggcgca tgaaggagtt

6961 tacatagaac ccgaagccag ggggcgcctg gatgctttga gagagtggat atactacaac

7021 tactacacag agcgagctaa gcgacgagac cggagacgca gatctgtttg tcacgcccgc

7081 acctggtttt gcttcaggaa atatgactac gtccggcgtt ccatttggca tgacactacg

7141 accaacacga tctcggttgt ctcggcgcac tccgtacagt agggatcgcc tacctccttt

7201 tgagacagag acccgcgcta ccatactgga ggatcatccg ctgctgcccg aatgtaacac

7261 tttgacaatg cacaacgtga gttacgtgcg aggtcttccc tgcagtgtgg gatttacgct

7321 gattcaggaa tgggttgttc cctgggatat ggttctgacg cgggaggagc ttgtaatcct 7381 gaggaagtgt atgcacgtgt gcctgtgttg tgccaacatt gatatcatga cgagcatgat

7441 gatccatggt tacgagtcct gggctctcca ctgtcattgt tccagtcccg gttccctgca

7501 gtgcatagcc ggcgggcagg ttttggccag ctggtttagg atggtggtgg atggcgccat

7561 gtttaatcag aggtttatat ggtaccggga ggtggtgaat tacaacatgc caaaagaggt

7621 aatgtttatg tccagcgtgt ttatgagggg tcgccactta atctacctgc gcttgtggta

7681 tgatggccac gtgggttctg tggtccccgc catgagcttt ggatacagcg ccttgcactg

7741 tgggattttg aacaatattg tggtgctgtg ctgcagttac tgtgctgatt taagtgagat

7801 cagggtgcgc tgctgtgccc ggaggacaag gcgtctcatg ctgcgggcgg tgcgaatcat

7861 cgctgaggag accactgcca tgttgtattc ctgcaggacg gagcggcggc ggcagcagtt

7921 tattcgcgcg ctgctgcagc accaccgccc tatcctgatg cacgattatg actctacccc

7981 catgtaggcg tggacttccc cttcgccgcc cgttgagcaa ccgcaagttg gacagcagcc

8041 tgtggctcag cagctggaca gcgacatgaa cttaagcgag ctgcccgggg agtttattaa

8101 tatcactgat gagcgtttgg ctcgacagga aaccgtgtgg aatataacac ctaagaatat

8161 gtctgttacc catgatatga tgctttttaa ggccagccgg ggagaaagga ctgtgtactc

8221 tgtgtgttgg gagggaggtg gcaggttgaa tactagggtt ctgtgagttt gattaaggta

8281 cggtgatcaa tataagctat gtggtggtgg ggctatacta ctgaatgaaa aatgacttga

8341 aattttctgc aattgaaaaa taaacacgtt gaaacataac atgcaacagg ttcacgattc

8401 tttattcctg ggcaatgtag gagaaggtgt aagagttggt agcaaaagtt tcagtggtgt

8461 attttccact ttcccaggac catgtaaaag acatagagta agtgcttacc tcgctagttt

8521 ctgtggattc actagaatcg atgtaggatg ttgcccctcc tgacgcggta ggagaagggg

8581 agggtgccct gcatgtctgc cgctgctctt gctcttgccg ctgctgagga ggggggcgca

8641 tctgccgcag caccggatgc atctgggaaa agcaaaaaag gggctcgtcc ctgtttccgg

8701 aggaatttgc aagcggggtc ttgcatgacg gggaggcaaa cccccgttcg ccgcagtccg

8761 gccggcccga gactcgaacc gggggtcctg cgactcaacc cttggaaaat aaccctccgg

8821 ctacagggag cgagccactt aatgctttcg ctttccagcc taaccgctta cgccgcgcgc

8881 ggccagtggc caaaaaagct agcgcagcag ccgccgcgcc tggaaggaag ccaaaaggag

8941 cgctcccccg ttgtctgacg tcgcacacct gggttcgaca cgcgggcggt aaccgcatgg

9001 atcacggcgg acggccggat ccggggttcg aaccccggtc gtccgccatg atacccttgc

9061 gaatttatcc accagaccac ggaagagtgc ccgcttacag gctctccttt tgcacggtct

9121 agagcgtcaa cgactgcgca cgcctcaccg gccagagcgt cccgaccatg gagcactttt

9181 tgccgctgcg caacatctgg aaccgcgtcc gcgactttcc gcgcgcctcc accaccgccg

9241 ccggcatcac ctggatgtcc aggtacatct acggattacg tcgacgttta aaccatatga

9301 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag

9361 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 9421 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg

9481 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg

9541 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga

9601 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc

9661 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt

9721 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact

9781 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg

9841 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt

9901 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt

9961 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct

10021 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg

10081 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt

10141 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt

10201 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc

10261 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg

10321 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc

10381 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg

10441 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca

10501 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga

10561 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct

10621 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg

10681 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca

10741 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata

10801 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct

10861 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact

10921 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa

10981 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc

11041 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga

11101 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga

11161 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt

11221 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag

11281 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga

11341 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg

11401 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc 11461 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 11521 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc 11581 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgatg gatcc

scAAV-CMV-luc2Pv2

LOCUS scAAV-CMV-luc2Pv 5968 bp DNA circular SYN 08-DEC-2014

REFERENCE 1 (bases 1 to 5968)

FEATURES Location/Qualifiers

misc_feature 1..106

/gene="mITR"

misc_feature 140..774

/gene="CMVpro"

CDS 806..2581

/gene="luc2P"

misc_feature 2668..2771

/note="3' ITR"

misc_feature 3319..3360

/note="Bacterial promoter"

misc_feature 3434..3702

/note="SV40 promoter"

misc_feature 3785..4579

/note="Neo/Kan"

misc_feature 4581..4833

/note="HSV tk poly A"

misc_feature 5325..5912

/note="pMBl ori"

ORIGIN (SEQ ID NO: 7)

1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt

61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

121 aggggttcct gcggccgcac gcgttgacat tgattattga ctagttatta atagtaatca

181 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta

241 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat

301 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga ctatttacgg

361 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac

421 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt 481 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg

541 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc

601 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt

661 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata

721 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg

781 actcactata gggagaccca agctcatgga agatgccaaa aacattaaga agggcccagc

841 gccattctac ccactcgaag acgggaccgc cggcgagcag ctgcacaaag ccatgaagcg

901 ctacgccctg gtgcccggca ccatcgcctt taccgacgca catatcgagg tggacattac

961 ctacgccgag tacttcgaga tgagcgttcg gctggcagaa gctatgaagc gctatgggct

1021 gaatacaaac catcggatcg tggtgtgcag cgagaatagc ttgcagttct tcatgcccgt

1081 gttgggtgcc ctgttcatcg gtgtggctgt ggccccagct aacgacatct acaacgagcg

1141 cgagctgctg aacagcatgg gcatcagcca gcccaccgtc gtattcgtga gcaagaaagg

1201 gctgcaaaag atcctcaacg tgcaaaagaa gctaccgatc atacaaaaga tcatcatcat

1261 ggatagcaag accgactacc agggcttcca aagcatgtac accttcgtga cttcccattt

1321 gccacccggc ttcaacgagt acgacttcgt gcccgagagc ttcgaccggg acaaaaccat

1381 cgccctgatc atgaacagta gtggcagtac cggattgccc aagggcgtag ccctaccgca

1441 ccgcaccgct tgtgtccgat tcagtcatgc ccgcgacccc atcttcggca accagatcat

1501 ccccgacacc gctatcctca gcgtggtgcc atttcaccac ggcttcggca tgttcaccac

1561 gctgggctac ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg aggaggagct

1621 attcttgcgc agcttgcaag actataagat tcaatctgcc ctgctggtgc ccacactatt

1681 tagcttcttc gctaagagca ctctcatcga caagtacgac ctaagcaact tgcacgagat

1741 cgccagcggc ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca aacgcttcca

1801 cctaccaggc atccgccagg gctacggcct gacagaaaca accagcgcca ttctgatcac

1861 ccccgaaggg gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa

1921 ggtggtggac ttggacaccg gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt

1981 ccgtggcccc atgatcatga gcggctacgt taacaacccc gaggctacaa acgctctcat

2041 cgacaaggac ggctggctgc acagcggcga catcgcctac tgggacgagg acgagcactt

2101 cttcatcgtg gaccggctga agagcctgat caaatacaag ggctaccagg tagccccagc

2161 cgaactggag agcatcctgc tgcaacaccc caacatcttc gacgccgggg tcgccggcct

2221 gcccgacgac gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac

2281 catgaccgag aaggagatcg tggactatgt ggccagccag gttacaaccg ccaagaagct

2341 gcgcggtggt gttgtgttcg tggacgaggt gcctaaagga ctgaccggca agttggacgc

2401 ccgcaagatc cgcgagattc tcattaaggc caagaagggc ggcaagatcg ccgtgaattc

2461 tcacggcttc cctcccgagg tggaggagca ggccgccggc accctgccca tgagctgcgc 2521 ccaggagagc ggcatggata gacaccctgc tgcttgcgcc agcgccagga tcaacgtcta

2581 aggccgcgac tctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact

2641 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg

2701 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg

2761 agcgagcgcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag

2821 ttgcgcagcc tgaatggcga atggaattcc agacgattga gcgtcaaaat gtaggtattt

2881 ccatgagcgt ttttcctgtt gcaatggctg gcggtaatat tgttctggat attaccagca

2941 aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat caaagaagta

3001 ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc ctcactgatt

3061 ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct ttaatcggcc

3121 tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg ctcgtcaaag

3181 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc

3241 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc

3301 tttctcgcca cgttcgccat cttcaaatat gtatccgctc atgagacaat aaccctgata

3361 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat

3421 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc

3481 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga

3541 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc

3601 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt

3661 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga

3721 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt

3781 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct

3841 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct

3901 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga

3961 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc

4021 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg

4081 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc

4141 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca

4201 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga

4261 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc

4321 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga

4381 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca

4441 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg

4501 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 4561 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc

4621 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga

4681 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc

4741 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc

4801 cgcgctatga cggcaataaa aagacagaat aaaaacgttg cgcaaactat taactggcga

4861 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc

4921 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc

4981 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg

5041 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat

5101 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata

5161 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct

5221 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga

5281 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg

5341 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc

5401 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct

5461 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc

5521 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt

5581 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg

5641 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct

5701 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 5761 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag

5821 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg

5881 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg

5941 gccttttgct cacatgtcct gcaggcag

pAAV-U6-sgRNA-uD

LOCUS pAAV-U6-sgRNA-uD 7141 bp DNA circular SYN 23-MAR-2017 DEFINITION pAAV-U6-sgRNA-uDys Circularized

FEATURES Location/Qualifiers

misc_feature 1..130

/gene="ITR"

misc_feature 162..3742

/note="huUDys"

misc_feature 3808..4039

/gene="bGH pA" misc_feature complement(4046..4126)

/gene="sgRNA scaffold"

misc_feature complement(4147..4395)

/gene="hU6"

misc_feature 4404..4544

/gene="ITR"

CDS 5461..6321

/gene="Amp"

misc_feature 6469..7136

/gene="pUC"

ORIGIN (SEQ ID NO: 8)

1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt

61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

121 aggggttcct gcggcctcta gactcgacat gggccgccac catgctgtgg tgggaggagg

181 tggaggattg ttatgaaagg gaggacgtgc agaagaagac ttttaccaag tgggtgaacg

241 ctcagttcag caaatttggg aagcagcaca tcgagaatct gttttccgac ctgcaggatg

301 ggagacggct gctggatctg ctggaaggac tgactggcca gaagctgccc aaagagaagg

361 ggagcactag ggtgcacgcc ctgaacaacg tgaacaaagc tctgagagtg ctgcagaaca

421 acaacgtgga tctggtgaat attggcagta ctgatatcgt ggacgggaac cacaaactga

481 cactgggcct gatctggaac attattctgc actggcaggt gaaaaatgtg atgaagaaca

541 tcatggccgg gctgcagcag accaattccg agaagatcct gctgtcttgg gtgcggcaga

601 gcacccgcaa ctatccccag gtgaacgtga ttaacttcac tacatcctgg agcgacgggc

661 tggccctgaa tgctctgatt cacagccaca ggcctgatct gttcgactgg aatagcgtgg

721 tgtgccagca gtctgccaca cagcgcctgg aacatgcctt caatatcgct cggtaccagc

781 tggggatcga aaaactgctg gacccagagg atgtggacac tacataccca gataaaaagt

841 ctattctgat gtacattact agcctgttcc aggtgctgcc acagcaggtg tctattgaag

901 ccattcagga ggtggaaatg ctgccccgcc cccccaaagt gactaaagag gagcattttc

961 agctgcatca tcagatgcat tacagccagc agattaccgt gagcctggct cagggatatg

1021 agcgcaccag tagtccaaaa ccacggttca agtcctacgc ttatacccag gctgcctacg

1081 tgacaactag cgaccctact agatccccct ttccatccca gcacctggag gccccagagg

1141 acaagagctt tgggtccagc ctgatggaaa gcgaggtgaa tctggatcgg taccagacag

1201 ccctggagga ggtgctgagc tggctgctga gtgctgaaga cacactgcag gcccagggcg

1261 aaatttccaa tgacgtggaa gtggtgaagg atcagttcca cacacacgag ggctatatga

1321 tggacctgac agctcaccag gggcgcgtgg gcaatatcct gcagctgggc tctaaactga 1381 tcggcaccgg gaaactgagt gaggacgagg aaacagaagt gcaggagcag atgaacctgc

1441 tgaacagccg ctgggagtgt ctgagagtgg ctagtatgga gaagcagtcc aacctgcacc

1501 gggtgctgat ggacctgcag aaccagaaac tgaaagagct gaacgactgg ctgacaaaga

1561 ctgaggaacg cacaaggaag atggaggagg agccactggg acccgacctg gaggatctga

1621 agagacaggt gcagcagcat aaggtgctgc aggaggatct ggaacaggag caggtgcggg

1681 tgaactccct gacacatatg gtggtggtgg tggacgaatc tagtggagat cacgccaccg

1741 ccgccctgga ggaacagctg aaggtgctgg gggaccggtg ggccaacatt tgccggtgga

1801 ccgaggacag gtgggtgctg ctgcaggaca tcctgctgaa atggcagagg ctgaccgagg

1861 agcagtgtct gtttagtgct tggctgagcg agaaagagga cgccgtgaac aagatccaca

1921 caaccggctt taaggatcag aacgaaatgc tgtctagcct gcagaaactg gctgtgctga

1981 aggccgatct ggagaaaaag aagcagagca tgggcaaact gtatagcctg aaacaggacc

2041 tgctgagcac cctgaagaac aagagcgtga cccagaagac agaagcctgg ctggataact

2101 ttgcccgctg ctgggacaac ctggtgcaga aactggagaa aagtacagct cagatctctc

2161 aggctgtgac cacaacccag cctagcctga cccagacaac cgtgatggaa accgtgacca

2221 ccgtgacaac ccgcgaacag atcctggtga aacatgccca ggaagagctg ccacctccac

2281 ctccccagaa gaagagaacc ctggagcggc tgcaggagct gcaggaagcc actgacgaac

2341 tggacctgaa gctgaggcag gccgaagtga ttaaggggtc ttggcagcct gtgggcgatc

2401 tgctgattga ttccctgcag gaccacctgg aaaaggtgaa ggctctgaga ggcgaaattg

2461 ctccactgaa ggagaacgtg agtcatgtga acgatctggc tagacagctg acaacactgg

2521 gcatccagct gagcccatac aatctgagca cactggagga cctgaatacc aggtggaagc

2581 tgctgcaggt ggctgtggaa gaccgggtgc ggcagctgca tgaggcccat cgcgacttcg

2641 gaccagccag ccagcacttt ctgagcacat ccgtgcaggg gccctgggag agggccattt

2701 ctcccaacaa ggtgccctac tatattaatc acgagaccca gaccacttgt tgggaccatc

2761 ccaagatgac agaactgtac cagtccctgg ccgatctgaa caacgtgagg tttagcgctt

2821 acagaaccgc tatgaagctg agacggctgc agaaggccct gtgcctggat ctgctgtccc

2881 tgtccgccgc ctgcgatgcc ctggatcagc ataatctgaa gcagaacgat cagccaatgg

2941 atatcctgca gatcatcaac tgcctgacca ctatctacga caggctggag caggagcaca

3001 acaacctggt gaacgtgcct ctgtgcgtgg atatgtgcct gaactggctg ctgaacgtgt

3061 atgacactgg gcgcaccggc cggatcagag tgctgagttt taaaactggg attatctccc

3121 tgtgtaaggc ccacctggag gacaagtaca ggtacctgtt caagcaggtg gctagtagca

3181 ctggattttg tgaccagcgc cgcctgggac tgctgctgca tgatagtatc cagattccta

3241 gacagctggg agaggtggct agtttcggag gatctaacat cgaacccagc gtgcgcagct

3301 gtttccagtt tgccaataac aaacctgaaa tcgaggctgc tctgttcctg gattggatgc

3361 gcctggaacc acagagcatg gtgtggctgc ctgtgctgca cagagtggct gccgccgaaa 3421 ctgccaagca ccaggctaaa tgcaacatct gcaaggaatg tcccattatc ggctttcgct

3481 acaggagtct gaaacatttt aactacgata tttgccagag ctgcttcttt tccggaagag

3541 tggccaaagg acacaagatg cactacccta tggtggaata ttgcacccca actacatctg

3601 gcgaagatgt gcgcgatttt gccaaggtgc tgaagaataa gtttcggact aagaggtact

3661 tcgccaagca cccccgcatg gggtatctgc cagtgcagac agtgctggaa ggagacaata

3721 tggagaccga tacaatgtga gcggccgcaa taaaagatct ttattttcat tagatctgtg

3781 tgttggtttt ttgtgtgtct agaattccta gagctcgctg atcagcctcg actgtgcctt

3841 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg

3901 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt

3961 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga

4021 atagcaggca tgctggggag gtaccaaaaa tctcgccaac aagttgacga gataaacacg

4081 gcattttgcc ttgttttagt agattctgtt tccagagtac taaaactgag acctgccgtg

4141 gtctccggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca

4201 agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc

4261 aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa

4321 attaattcca attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat

4381 gggaaatagg ccctcgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct

4441 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc

4501 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta

4561 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac

4621 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct

4681 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg

4741 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt

4801 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca

4861 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga

4921 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa

4981 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac

5041 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc

5101 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga

5161 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc

5221 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata

5281 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact

5341 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg

5401 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 5461 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct

5521 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca

5581 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc

5641 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc

5701 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg

5761 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta

5821 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc

5881 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt

5941 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg

6001 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct

6061 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc

6121 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc

6181 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac

6241 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc

6301 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat

6361 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg

6421 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc

6481 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa

6541 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag

6601 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta

6661 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta

6721 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag

6781 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg

6841 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg

6901 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag

6961 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc

7021 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa

7081 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg

7141 1

Spacer (SEQ ID NO: 9)

gcctccaagacccagagcattaagaagtacagcacagacattctgggcaacctgtatgaagtgaaatctaagaagcaccctcagatca tcaaaaagggcagcggcttcgccaacgagctgggccctagactgatgggaaagactagtagaccggtagagccatcaccccagcg Dmd gRNA 1 (SEQ ID NO: 10)

ATATAATAGAAATTATTCAT

Dmd gRNA 2 (SEQ ID NO: 11)

TAATATGCCCTGTAATATAA

Dmd gRNA 3 (SEQ ID NO: 12)

TGATATCATCAATATCTTTG

Dmd gRNA 4 (SEP ID NO: 13)

GC AATTAATTGGAAA ATGTG

Dmd gRNA 5 (SEQ ID NO: 14)

CTTTAAGCTTAGGTAAAATCA

Dmd gRNA 6 (SEQ ID NO: 15)

CAGTAATGTGTCATACCTTC

Dmd gRNA 7 (SEQ ID NO: 16)

CAGGGCATATTATATTTAGA

Dmd gRNA 8 (SEQ ID NO: 17)

CAAAAGCCAAATCTATTTCA

spCas9 (SEQ ID NO: 18)

>sp|Q99ZW2|CAS9_STRPl CRISPR-associated endonuclease Cas9/Csnl OS=Streptococcus pyogenes serotype Ml GN=cas9 PE=1 SV=1

MDKXYSIGLDIGTOSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE

TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE

RHPIFGNIVDEVAYHE YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG

DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA AILSARLSKSRRLENLIAQLP

GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA

DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE

KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR

TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA

WMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV

YNELTKVK TEGMRKPAFLSGEQKKAIVDLLF TNRKVTVKQLKEDYFKKIECFD

SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL

KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD QSGKTILDFLKSDGFANRN

FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK

VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL

QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMK YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK

RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

REIN YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK

ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM

PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV

AKVEKGKSKKLKSVKELLGITIMERSSFEK PIDFLEAKGYKEVKKDLIIKLPKYSLFE

LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ

HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA

PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

Cpfl (SEQ ID NO: 19)

CPFI FRATN CRISPR-associated endonuclease Cpfl OS=Francisella tularensis subsp. novicida (strain U112) GN=cpfl PE=1 SV=1

MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYH

QFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSE

KFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT

TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK

KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGEN

TKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTM

QSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY

SVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI

DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIK

DLLDQTTslNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYI

TQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFD

DKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN

GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE

NQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDER

NLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKR

FTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDG

KGNnKQDTFNnGNDRMKTOYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQV

VHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEF

DKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESV

SKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKN

HNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQM RNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRI

KNNQEGKKLNLVIKNEEYFEFVQNRNN

SpCas9 PAM (SEQ ID NO: 20)

NGG

SpCas9 D1135E variant PAM (SEQ ID NO: 21)

NGG

S^pCas9 VRER variant PAM (SEQ ID NO: 22)

NGCG

SpCas9 EOR variant PAM (SEQ ID NO: 23)

NGAG

SpCas9 VOR variant PAM 1 (SEQ ID NO: 24)

NGAN

S^pCas9 VOR variant PAM 1 (SEQ ID NO: 25)

NGNG

SaCas9 PAM 1 (SEQ ID NO: 26)

NNGRRT

SaCas9 PAM 2 (SEQ ID NO: 27)

NNGRR

NMCas9 PAM (SEQ ID NO: 28)

NNNNGATT

STCas9 PAM (SEQ ID NO: 29)

NNAGAAW

TP Cas9 PAM (SEQ ID NO: 30)

NAAAAC

Linker 1 (SEQ ID NO: 31)

KESGSVSSEQLAQFRSLD

Linker 1 (SEQ ID NO: 32)

EGKSSGSGSESKST

Linker 1 (SEQ ID NO: 33)

GGGGGGGG

Linker 1 (SEQ ID NO: 34)

GSAGSAAGSGEF

Linker 1 (SEQ ID NO: 35)

AEAAAK saCas9-VP2 fusion peptide (SEQ ID NO: 36)

(pNLRep2-Caprh74-AVB-VP2NN-VP3knock-Cas9update starting at bp 5532)

MAPKKKRKVGIHGVPAAKRNYILGLDIGITSVGYGIIDYETRDVIDAGVR

LFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELS

GINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELST

KEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL

KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLM

GHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQII

ENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKD

ITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQIS

NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQK

EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNS

KDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKC

LYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKG

NRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINR

FSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFL

RRKWKFKKERNKGYKHHAEDALnANADFIFKEWKKLDKAKKVMENQMFE

EKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELI

NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDP

QTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYG

NKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVI

KKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGV

NNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDI

LGNLYEVKSKKHPQIIKKGSGFANELGPRLMGKTSTAPGKKRPVEPSPQR

SPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSG

TLAAGGGAPLADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPT

YNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRL

INNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQ

LPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFP

SQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQ

STGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSN

FAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGK

DNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQG

ALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIK NTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQ

YTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

VP1 protein (SEQ ID NO: 37)

Translation of VP1 (pAAVrh74-VPl-3 starting at bp 5037)

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGY

KYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEF

QERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKAAPGKKRPVEPSP

QRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLG

SGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWAL

PTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ

RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSE

YQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEY

FPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSR

TQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNN

SNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGA

GKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS

QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQIL

IKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPE

IQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

VP3 protein (SEQ ID NO: 38)

Translation of VP3 (pAAVrh74-VPl-3 starting at bp 5646)

MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTY

NNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLI

NNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQL

PYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPS

QMLRTGNNFEF S YNFED VPFHS S Y AHS Q SLDRLMNPLID QYL YYL SRTQ S

TGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNF

AWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKD

NVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGA

LPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKN

TPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQY

TSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

VP2 protein (SEQ ID NO: 39)

Translation of VP2 (pNLRep2-Caprh74-AVB-VP2 starting at bp 5448) MASGKKRSVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQ

PIGEPPAGPSGLGSGTMAAGGGAPMADN EGADGVGSSSGNWHCDSTWLG

DRVITTSTRTWALPTYN HLYKQISNGTSGGSTNDNTYFGYSTPWGYFDF

NRFHCHFSPRDWQRLINN WGFRPKRLNFKLFNIQVKEVTQNEGTKTIAN

NLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN GS

QAVGRSSFYCLEYFPSQMLRTGN FEFSYNFEDVPFHSSYAHSQSLDRLM

NPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPN MSAQAK WLPGPCYR

QQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFF

PSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQ

QQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLM

GGFGLKHPPPQILIK TPVPADPPTTFSQAKLASFITQYSTGQVSVEIEW

ELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

dSaCas9 protein (SEQ ID NO: 40)

Translation of dSaCas9 (pX603-AAV-CMV-NLS-dSaCas9-NLS-3xHA-bGHpA starting at bp 700)

MGGRRVRWEVYISRALWLTTGATMAPKKKRKVGIHGVPAAKRNYILGLAI

GITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRR

HRIQRVKKLLFD YNLLTDHS EL S GINP YEARVKGLS QKL SEEEF S AALLH

LAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKD

GEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTY

YEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALND

LNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIK

GYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSS

EDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTN

DNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVI

NAnKKYGLPNDniELAP^KNSKDAQKMINEMQKRNRQTNERIEEIIRT

TGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR

SVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNL

AKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLR

SYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANA

DFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIK

HIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDK

DNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET

GNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKP YRFDVYLDNGVYKFVTVK LDVIKKENYYEVNSKCYEEAKKLKKISNQAE

FIASFYN DLIKINGELYRVIGVN DLLNRIEVNMIDITYREYLENMNDK

RPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGKRPAATKK

AGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA

CMV promoter (SEQ ID NO: 41)

TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA

CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA

CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT

ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG

GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTAC

GTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC

GTGGAT AGC GGTTTGACTC AC GGGGATTTC C AAGTCTCC ACCC C ATTGAC GTC AA

TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAAC

TCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATA

AGCAGAGCTGGTTTAGTGAACCGTCAG

U6 promoter (SEQ ID NO: 42)

ggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca

agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc

aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa

attaattcca attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat

gggaaatagg ccctc

Claims

What is claimed is:

1. A modified viral capsid protein comprising a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior of the viral capsid protein.

2. The modified capsid protein of claim 1, further comprising a spacer.

3. The modified capsid protein of claim 1, wherein the viral capsid is selected from the group of an adenoviral (Ad) capsid protein or an adeno-associated virus (AAV) capsid protein.

4. The modified capsid protein of claim 1, wherein AAV viral protein comprises one or more of VP1, VP2, and VP3, or an equivalent of each thereof.

5. The modified viral capsid protein of claim 4, wherein the AAV viral protein comprises VP2, or an equivalent thereof.

6. The modified viral capsid protein of any one of claims 1-5, wherein the Cas9 protein is a S. aureus Cas 9.

7. The modified viral capsid protein of claim 6, wherein the modified capsid protein comprises SEQ ID NO: 36, or an equivalent thereof.

8. An isolated polynucleotide encoding the modified capsid protein of claim 5.

9. An isolated polynucleotide encoding the modified capsid protein of claim 6.

10. A vector or host cell comprising the isolated polynucleotide of claim 7.

11. A vector or host cell comprising the isolated polynucleotide of claim 8.

12. A vector or host cell comprising the isolated polynucleotide of claim 9.

13. A method of preparing the capsid protein of any of claims 1 to 5, comprising coupling the Cas9 protein or an equivalent thereof to the viral capsid protein.

14. A method of preparing the capsid protein of claim 6, comprising coupling the Cas9 protein or an equivalent thereof to the viral capsid protein.

15. A method of preparing the capsid protein of claim 7, comprising coupling the Cas9 protein or an equivalent thereof to the viral capsid protein.

16. A method of preparing a modified capsid protein comprising expressing the polynucleotide of claim 8.

17. A method of preparing a modified capsid protein comprising expressing the polynucleotide of claim 9.

18. A method of preparing a modified capsid protein comprising expressing the polynucleotide of claim 10.

19. A recombinant viral particle comprising the modified capsid of any one of claims 1 to 5, and a polynucleotide encapsulated within the capsid.

20. A recombinant viral particle comprising the modified capsid of claim 6 and a polynucleotide encapsulated within the capsid.

21. A recombinant viral particle comprising the modified capsid of claim 7 and one or more polynucleotides encapsulated within the capsid.

22. The recombinant viral particle of claim 19, wherein at least one of the polynucleotide comprises a polynucleotide encoding a guide RNA (gRNA).

23. The recombinant viral particle of claim 22, wherein the polynucleotide encoding the gRNA comprises:

a. a fusion polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or

b. a polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).

24. The recombinant viral particle of claim 21, further comprising a therapeutic polynucleotide.

25. The recombinant viral particle of claim 24, wherein the therapeutic polynucleotide comprises a repair template.

26. The recombinant viral particle of claim 20, wherein at least one of the polynucleotide comprises a polynucleotide encoding a guide RNA (gRNA).

27. The recombinant viral particle of claim 26, wherein the polynucleotide encoding the gRNA comprises:

a. a fusion polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or

b. a polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).

28. The recombinant viral particle of claim 22, further comprising a therapeutic polynucleotide.

29. The recombinant viral particle of claim 28, wherein the therapeutic polynucleotide comprises a repair template.

30. The recombinant viral particle of claim 21, wherein at least one of the polynucleotide comprises a polynucleotide encoding a guide RNA (gRNA).

31. The recombinant viral particle of claim 30, wherein the polynucleotide encoding the gRNA comprises:

a. a fusion polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or

b. a polypeptide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).

32. The recombinant viral particle of claim 30, further comprising a therapeutic polynucleotide.

33. The recombinant viral particle of claim 32, wherein the therapeutic polynucleotide comprises a repair template.

34. A recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the viral particle surface, comprising:

(a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or the equivalent thereof and a viral capsid protein; and

(b) a helper plasmid.

35. The recombinant expression system of claim 34, wherein the viral capsid is selected from the group of an adenoviral (Ad) capsid protein or an adeno-associated virus (AAV) capsid protein.

36. The modified viral capsid of claim 35, wherein AAV viral protein comprises one or more of VP1, VP2, and VP3, or an equivalent of each thereof.

37. The modified viral capsid of claim 36, wherein the AAV viral protein comprises VP2, or an equivalent thereof.

38. The modified viral capsid of any one of claims 34-37, wherein the Cas9 protein is a S. aureus Cas 9.

39. The modified viral capsid of claim 38, wherein the Cas9 protein comprises SEQ ID NO: 3, or an equivalent thereof.

40. The recombinant expression system of claim 34, wherein the fusion protein comprises Cas9 and VP2.

41. The recombinant expression system of any one of claims 34-40, wherein plasmid (a) comprises a DNA sequence selected from the group of a DNA sequence encoding VP2, a DNA sequence encoding Cas9, a DNA sequence encoding SEQ ID NO: 36, or an equivalent of each thereof.

42. The recombinant expression system of any one of claims 34-40, wherein helper plasmid (b) comprises a DNA sequence selected from the group of a DNA sequence encoding VPl, a DNA sequence encoding VP3, or a DNA sequence encoding both VPl and VP3, or an equivalent of each thereof.

43. The recombinant expression system of claim 42, wherein the helper plasmid comprises SEQ ID NO: 6 or an equivalent thereof.

44. The recombinant expression system of any one of claims 34-40, further comprising a polynucleotide encoding one or more guide RNAs.

45. The recombinant expression system of any one of claims 34-40, further comprising a therapeutic polynucleotide.

46. The recombinant expression system of claim 44, further comprising a therapeutic polynucleotide.

47. A method of producing modified AAV expressing Cas9 on its surface comprising transfecting one or more cells with the recombinant expression system of any one of claims 40-44.

48. A method of producing modified AAV expressing Cas9 on its surface comprising transfecting one or more cells with the recombinant expression system of claim 45.

49. A method of producing modified AAV expressing Cas9 on its surface comprising transfecting one or more cells with the recombinant expression system of claim 46.

50. A modified AAV produced according to the method of claim 47.

51. A modified AAV produced according to the method of claim 48.

52. A modified AAV produced according to the method of claim 49.

53. An isolated tissue comprising the modified viral particle of any one of claims 1 -5.

54. An isolated tissue comprising the modified viral particle of claim 6.

55. An isolated tissue comprising the modified viral particle of claim 7.

56. A non-human transgenic animal comprising the modified viral particle of any one of claims 1-5.

57. A non-human transgenic animal comprising the modified viral particle of claim 6.

58. A non-human transgenic animal comprising the modified viral particle of claim 7.

59. A method of gene editing comprising contacting a cell with the recombinant viral particle of claim 22.

60. A method of gene editing comprising contacting a cell with the recombinant viral particle of claim 24.

61. The method of claim 59, wherein the contacting is in vitro or in vivo.

62. The method of claim 60, wherein the contacting is in vitro or in vivo.

63. A method of gene editing in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant viral particle of claim 22.

64. A method of gene editing in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant viral particle of claim 24.

65. The method of claim 64, wherein the therapeutic polynucleotide is selected to treat a disease, disorder, or condition selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha- 1 -antitrypsin, amyotrophic lateral sclerosis, Alzheimer's, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson's, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesteremia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysarccharidosis (I-IX), hepatitis B, and hepatitis C.

66. The method of claim 65, wherein the hemophilia is characterized by one or more of factor VIII or factor IX deficiency.

67. The method of claim 65, wherein the muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery -Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.