Therapeutic applications of CPF1-based genome editing

Abstract

To provide therapeutic applications of CRISPR / Cpf1-based genome editing. [Solution] The present invention provides a Cpf1 guide RNA (gRNA) that targets the dystrophin gene and includes a polynucleotide sequence having a specific sequence or its complement, and a DNA targeting composition that includes a Cpf1 endonuclease and the Cpf1 gRNA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 62 / 363,888, filed July 19, 2016, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy above was created on July 19, 2017, is referred to as 028193-9250-WO00SequenceListing.txt, and is 46,056 bytes in size.

[0003] STATEMENT OF GOVERNMENT RIGHTS This invention was made with Government support under Federal Grant Nos. AR069085 and MD140071 awarded by the NIH and Army / MRMC, respectively. The United States Government has certain rights in the invention.

[0004] The present disclosure relates to the fields of altered gene expression, genomic engineering of genes, and genomic modification using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR / Cpf1) based systems and viral delivery systems from Prevotella and Francisella 1. [Background technology]

[0005] RNA-guided nucleases have been engineered for genome modification in human cells, such as the CRISPR / Cpf1 system from Streptococcus pyogenes and Staphylococcus aureus. Many microorganisms have been found to have DNA editing or RNA editing systems. Cas9 from S.pyogenes and S.aureus causes blunt-end double-strand breaks (DSBs) in genomic DNA, which are repaired by non-homologous end joining (NHEJ), which leaves small insertions and deletions (indels) at the repair site, or by homologous recombination repair in the presence of a template. These indels can be used to knock out genes, remove splice acceptors, or cleave gene regulatory elements.

[0006] Inherited genetic diseases have a devastating impact on children in the United States. These diseases currently have no cure and can only be managed by attempting to alleviate symptoms. For decades, the field of gene therapy has promised a cure for this disease. However, technical obstacles to the safe and efficient delivery of therapeutic genes to cells and patients have limited this approach. Duchenne muscular dystrophy (DMD) is a fatal genetic disease clinically characterized by muscle wasting, loss of ambulation, and death typically in the third decade of life due to the loss of functional dystrophin. DMD is the result of inherited or spontaneous mutations in the dystrophin gene. Most mutations causing DMD are the result of exon deletions, pushing the translation reading frame out of frame.

[0007] Dystrophin is a key component of a protein complex involved in regulating muscle cell integrity and function. DMD patients generally lose the ability to physically support themselves during childhood, become increasingly weak during their teenage years, and die during their twenties. Current experimental gene therapy strategies for DMD require repeated administration of transient gene delivery vehicles or rely on the permanent integration of foreign genetic material into genomic DNA. Both of these methods have serious safety concerns. In addition, these strategies are limited by their inability to deliver the large and complex dystrophin gene sequence. There is a need for more precise and efficient gene editing tools to correct or treat patients with mutations in the dystrophin gene. Summary of the Invention [Means for solving the problem]

[0008] The present invention is directed to a Cpf1 guide RNA (gRNA) that targets the dystrophin gene and comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

[0009] The present invention is directed to a DNA targeting composition comprising a Cpf1 endonuclease and at least one Cpf1 gRNA as described herein.

[0010] The present invention is directed to a DNA targeting composition comprising a first Cpf1 gRNA and a second Cpf1 gRNA, wherein the first Cpf1 gRNA and the second Cpf1 gRNA each comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and wherein the first Cpf1 gRNA and the second Cpf1 gRNA target the dystrophin gene.

[0011] The present invention is directed to an isolated polynucleotide comprising a polynucleotide sequence encoding the Cpf1 gRNA described above or the DNA targeting composition described above.

[0012] The present invention is directed to a vector comprising a polynucleotide sequence encoding the Cpf1 gRNA described above, a DNA targeting composition described above, or an isolated polynucleotide described above.

[0013] The present invention is directed to a vector encoding (a) a first Cpf1 guide RNA (gRNA), (b) a second Cpf1 gRNA, and (c) at least one Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences.

[0014] The present invention is directed to a cell comprising the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, an isolated polynucleotide described above, or a vector described above.

[0015] The present invention is directed to a kit comprising the Cpf1 gRNA described above, a polynucleotide sequence encoding the DNA targeting composition described above, an isolated polynucleotide described above, a vector described above, or a cell described above.

[0016] The present invention is directed to a composition for deleting a segment including exon 51 in a dystrophin gene, the composition comprising: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 The gRNA comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and the first vector and the second vector are configured to form first and second double-stranded breaks in a first intron and a second intron, respectively, adjacent to exon 51 of the human DMD gene, thereby deleting a segment including exon 51 in the dystrophin gene.

[0017] The present invention is directed to a cell comprising the composition described above.

[0018] The present invention is directed to a modified adeno-associated viral vector for genome editing of a mutant dystrophin gene in a subject, the modified adeno-associated viral vector comprising a first polynucleotide sequence encoding a Cpf1 gRNA as described above, and a second polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0019] The present invention is directed to a method of correcting a mutated dystrophin gene in a cell, the method comprising administering to the cell a polynucleotide sequence encoding a Cpf1 gRNA as described above, a DNA targeting composition as described above, an isolated polynucleotide as described above, a vector as described above, a composition as described above, or a modified adeno-associated viral vector as described above.

[0020] The present invention is directed to a method of genome editing a mutant dystrophin gene in a subject, the method comprising administering to the subject a genome editing composition comprising a polynucleotide sequence encoding a Cpf1 gRNA as described above, a DNA targeting composition as described above, an isolated polynucleotide as described above, a vector as described above, a composition as described above, or a modified adeno-associated viral vector as described above.

[0021] The present invention is directed to a method of treating a subject in need thereof having a mutated dystrophin gene, the method comprising administering to the subject a polynucleotide sequence encoding a Cpf1 gRNA as described above, a DNA targeting composition as described above, an isolated polynucleotide as described above, a vector as described above, a composition as described above, or a modified adeno-associated viral vector as described above.

[0022] The present invention is directed to a method of correcting a mutant dystrophin gene in a cell, comprising administering to the cell (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 The gRNA comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the vector is configured to form first and second double-stranded breaks in first and second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene to correct a mutant dystrophin gene in the cell.

[0023] The present invention is directed to a method of treating a subject in need thereof having a mutated dystrophin gene, the method comprising administering to the subject (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 The gRNA comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first vector and the second vector are configured to form first and second double-stranded breaks in first and second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene to treat the subject.

[0024] The present invention is directed to a Cpf1 guide RNA (gRNA) that targets an enhancer of the B-cell lymphoma / leukemia 11A (BCL11a) gene and comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 65-70, or a complement thereof.

[0025] The present invention is directed to a method for disrupting an enhancer of the B-cell lymphoma / leukemia 11A gene in a cell, the method comprising administering to the cell at least one Cpf1 gRNA and Cpf1 endonuclease as described above. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a schematic diagram showing the use of Cpf1 in three treatments for genetic diseases such as DMD and SCA / β-thalassemia, according to some embodiments of the present disclosure. [Diagram 2] 1 shows a blot revealing that exons 44, 46 and 51 are target gRNAs with detectable activity according to some embodiments of the present disclosure. [Diagram 3] 1 shows a blot demonstrating that 42 guide RNA pairs targeting exon 51 deletions were screened according to one embodiment of the present disclosure. [Figure 4] We show that SaCas9 and LbCpf1 are expressed in patient-derived myoblasts. [Diagram 5] 1 shows genomic deletions caused by SaCas9 or LbCpf1 in patient-derived myoblasts. [Figure 6] We show that SaCas9 or LbCpf1 targeted exon 51 deletion removes the exon from the transcript. [Figure 7] A population of Cpf1 crRNAs revealing surveyor nuclease activity throughout the exon is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present disclosure provides, in part, therapeutic applications of CRISPR / Cpf1-based genome editing for treating disease. Cpf1, a V-type CRISPR-Cas effector endonuclease, is involved in adaptive immunity in prokaryotes, including Acidaminococcus and Lachnospiraceae, among others, and exhibits gene editing activity in human cells through a single RNA-guided approach. The present disclosure provides methods that can use CRISPR / Cpf1-based systems to treat genetic diseases, such as Duchenne muscular dystrophy (DMD), sickle cell anemia (SCA) and β-thalassemia.

[0028] According to one embodiment of the present disclosure, the first method includes splice acceptor knockout. Cpf1 generates a relatively large indel footprint, which makes the destruction of splice acceptors and the removal of target exons from transcripts efficient (see FIG. 1A). As shown in FIG. 1A, Cpf1 causes a 5-base pair staggered double-strand break from DNA (which can be repaired via non-homologous end joining (NHEJ)), producing a relatively large insertion or deletion (indel) footprint, followed by S. pyogenes or S. aureus Cas9. This allows for more powerful destruction of splice acceptors and even removal of target exons, since repair leaves a relatively large indel footprint, which can make the knockout of genetic elements such as splice acceptors and enhancers more efficient. Cpf1 also has a distinct protospacer adjacent motif (PAM) that increases the diversity of genomic regions that can be targeted. Cpf1 recognizes TTTN, whereas S. pyogenes Cas9 recognizes NGG and S. aureus Cas9 recognizes NNGRRT. Furthermore, Cpf1 does not require tracrRNA, and thus only crRNA is required, and therefore also uses small guide RNA.

[0029] Another aspect of the present disclosure provides a method that includes compatible overhang deletion. Cpf1 can facilitate gene deletion by matching overhangs to remove genetic elements (see FIG. 1B). As shown in FIG. 1B, Cpf1 produces a 5 base pair overhang that can be compatible with a second double-stranded break. Multiple Cpf1 guide RNAs can be provided with compatible overhangs to facilitate seamless gene deletion. Previous studies with S. aureus Cas9 have demonstrated that approximately 67% of gene deletions are seamless for one guide RNA pair. For example, compatible overhangs produced by multiplexing Cpf1 around a genetic region of interest (e.g., exon 51 in dystrophin) can facilitate seamless deletion. After NHEJ, a gene deletion is achieved that can restore the reading frame of the mutated gene. Matching overhangs can facilitate very precise ligation.

[0030] Another aspect of the present disclosure provides a method comprising enhancer disruption. Cpf1 can produce a relatively large indel footprint that makes the disruption of enhancers and other gene regulatory elements more reliable (see Fig. 1C). As shown in Fig. 1C, the relatively large indel footprint produced by Cpf1 can also be exploited to disrupt enhancers for investigating enhancer function, or as potential treatments for diseases such as SCA.

[0031] For example, the present disclosure describes the modification of Cpf1 for targeted gene removal of single and multiple exons of the dystrophin gene for the treatment of Duchenne muscular dystrophy (DMD). This is achieved by targeted mutagenesis of splice acceptors at mutation hotspots for single exon removal, or gene deletion of single or multiple exons. Targeted exon removal can restore the reading frame of dystrophin, resulting in improved muscle function and patient phenotype. Gene enhancers can also be targeted as a therapeutic approach to disease treatment, particularly the BCL11a enhancer region or gamma globin promoter as a treatment for sickle cell anemia (SCA) or beta thalassemia. To restore functional dystrophin expression in cells from DMD patients, the disclosed Cpf1 gRNA can be used together with a CRISPR / Cpf1-based system to target gene regions, such as the intronic region around exon 51 of the human dystrophin gene, to cause gene deletion in this region.

[0032] Also described herein are gene constructs, compositions and methods for delivering a gene editing system based on CRISPR / Cpf1 and multiple gRNAs to target dystrophin gene.The subject matter of the present disclosure also provides a method for delivering a gene construct (e.g., vector) or a composition comprising this gene construct to skeletal muscle.This vector can be AAV (e.g., modified AAV vector).The subject matter of the present disclosure describes a method for delivering the active form of this class of therapeutic agent to skeletal muscle that is effective, efficient and facilitates successful genome modification, and further provides a means to rewrite the human genome for therapeutic applications and a target model species for basic science applications.

[0033] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0034] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, will control. Although preferred methods and materials are described below, similar or equivalent methods and materials to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0035] The terms "comprise," "including," "having," "having," "can," "containing," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude additional actions or structural possibilities. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements provided herein, whether or not expressly described.

[0036] For the recitation of numerical ranges herein, each intervening number is specifically contemplated to the same degree of precision. For example, for the range 6 to 9, the numbers 7 and 8 are specifically contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated.

[0037] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 3 or more than 3 standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, preferably within a range of up to 10%, more preferably within a range of up to 5%, and even more preferably within a range of up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0038] "Adeno-associated virus" or "AAV", as used interchangeably herein, refers to a small virus belonging to the Dependovirus genus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease, and therefore the virus induces a very mild immune response.

[0039] As used herein, "binding region" refers to the region within a nuclease target region that is recognized and bound by a nuclease.

[0040] "Cardiomyocyte" or "heart muscle", as used interchangeably herein, refers to a type of involuntary striated muscle (myocardium) found in the wall and histological basis of the heart. Cardiac muscle is made up of cardiomyocytes or myocardiocytes. Cardiomyocytes exhibit striations similar to those on skeletal muscle cells, but unlike multinucleated skeletal muscle, contain only one unique nucleus. In certain embodiments, "myocardial condition" refers to a condition associated with the myocardium (e.g., cardiomyopathies, heart failure, arrhythmias, and inflammatory heart disease).

[0041] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) that comprises a polynucleotide sequence that codes for a protein. The coding sequence may further comprise start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0042] As used herein, "complement" or "complementary" means that a nucleic acid can undergo Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between polynucleotides or polynucleotide analogs of a nucleic acid molecule. "Complementarity" refers to the property shared between two nucleic acid sequences such that when aligned antiparallel to each other, the polynucleotide bases at each position are complementary.

[0043] As used herein, "correcting", "genome editing" and "repairing" refer to changing a mutant gene that codes for a truncated protein or no protein at all to obtain full-length functional or partially full-length functional protein expression. Correcting or repairing a mutant gene can include replacing a region of the gene that has a mutation, or replacing the entire mutant gene with a copy of the gene that does not have the mutation using a repair mechanism such as homology-directed repair (HDR). Correcting or repairing a mutant gene can also include repairing a frameshift mutation that results in a premature stop codon, an ectopic splice acceptor site, or an ectopic splice donor site by creating a double-strand break in the gene and then repairing the gene using non-homologous end joining (NHEJ). NHEJ can add or delete at least one base pair during repair, which can restore the proper reading frame and remove the premature stop codon. Correcting or repairing a mutant gene can also include destroying the ectopic splice acceptor site or splice donor sequence. Correcting or repairing a mutated gene can also include deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand to restore the proper reading frame by removing the DNA between two nuclease target sites, and repairing the DNA break by NHEJ.

[0044] "Cpf1 endonuclease" or "Cpf1", as used interchangeably herein, refers to a single RNA-guided endonuclease of the Class 2 CRISPR-Cas system that is a smaller and simpler endonuclease than Cas9. The Cpf1 endonuclease targets and cleaves as a 5-nucleotide staggered cut distal to the 5' T-rich PAM.

[0045] "Donor DNA," "donor template," and "repair template," as used interchangeably herein, refer to a double-stranded DNA fragment or molecule that contains at least a portion of a gene of interest. The donor DNA can encode a fully functional protein or a partially functional protein.

[0046] "Duchenne muscular dystrophy" or "DMD", used interchangeably herein, refers to a recessive, lethal, X-linked disorder that results in muscle degeneration and ultimately death. DMD is a common, inherited, monogenic disease that affects 1 in 3500 males. DMD is the result of inherited or spontaneous mutations that result in nonsense or frameshift mutations in the dystrophin gene. The majority of dystrophin mutations that cause DMD are exon deletions that disrupt the reading frame in the dystrophin gene, causing premature translation termination. DMD patients typically lose the ability to support themselves during childhood, develop progressive muscle weakness during their teenage years, and die in their twenties.

[0047] "Dystrophin" as used herein refers to a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix through the cell membrane. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane, which is responsible for regulating muscle cell integrity and function. The dystrophin gene or "DMD gene", as used interchangeably herein, is 2.2 megabases at the locus Xp21. The primary transcript is approximately 2,400 kb, and the mature mRNA is approximately 14 kb. 79 exons code for a protein that is more than 3500 amino acids.

[0048] "Exon 51" as used herein refers to the 51st exon of the dystrophin gene. Exon 51 is frequently adjacent to frame-breaking deletions in DMD patients and has been targeted in clinical trials for oligonucleotide-based exon skipping. A clinical trial for the exon 51 skipping compound eteplirsen recently reported significant functional benefit over 48 weeks, with an average of 47% dystrophin-positive fibers compared to baseline. Mutations in exon 51 are ideally suited for permanent correction by NHEJ-based genome editing.

[0049] As used herein interchangeably, "frameshift" or "frameshift mutation" refers to a type of genetic mutation in which the addition or deletion of one or more polynucleotides results in a shift in the reading frame of a codon in an mRNA. The shift in reading frame may result in a change in the amino acid sequence in protein translation, such as a missense mutation or a premature stop codon.

[0050] As used herein, "functional" and "fully functional" describe a protein that has biological activity. A "functional gene" refers to a gene that is transcribed into mRNA that is translated into a functional protein.

[0051] "Genetic construct" as used herein refers to a DNA or RNA molecule that comprises a polynucleotide sequence that codes for a protein.The coding sequence comprises start and stop signals that are operably linked to regulatory elements, including promoters and polyadenylation signals, that can induce expression in the cells of the individual to which the nucleic acid molecule is administered.As used herein, the term "expressible form" refers to a genetic construct that contains the necessary regulatory elements that are operably linked to the coding sequence that codes for a protein, such that the coding sequence is expressed when present in the cells of the individual.

[0052] As used herein, "genetic disease" refers to a disease, particularly a condition present from birth, caused in part or in whole, directly or indirectly, by one or more abnormalities in the genome. The abnormality may be a mutation, insertion, or deletion. The abnormality may affect the coding sequence of a gene or its regulatory sequence. The genetic disease may be, but is not limited to, DMD, Becker muscular dystrophy (BMD), hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of liver metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia such as beta thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease.

[0053] " Homologous recombination repair " or " HDR ", used interchangeably herein, refers to the mechanism in cells to repair double-stranded DNA damage when homologous fragments of DNA are present in the nucleus, usually in the G2 and S phases of the cell cycle. HDR uses donor DNA template to guide repair, and can be used to create specific sequence changes to genomes, including targeted addition of whole genes. When donor template is provided with CRISPR / Cpf1-based gene editing system, the cellular machinery will repair the break by homologous recombination, which is enhanced by several orders of magnitude in the presence of DNA break. In the absence of homologous DNA fragments, non-homologous end joining can occur instead.

[0054] "Genome editing" as used herein refers to changing genes. Genome editing can include correcting or repairing mutated genes. Genome editing can include knocking out genes, such as mutated or normal genes. Genome editing can be used to treat disease or enhance muscle repair by changing targeted genes.

[0055] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are identical over a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions where identical residues exist in both sequences to give the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and multiplying the result by 100 to give the percentage of sequence identity. If the two sequences are of different lengths, or the alignment results in one or more sticky ends such that only a single sequence is included in a particular region of comparison, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0056] As used herein interchangeably, "mutated gene" or "mutated gene" refers to a gene that has undergone a detectable mutation. A mutant gene has undergone an alteration, such as loss, gain, or exchange of genetic material, that affects the normal transmission and expression of the gene. As used herein, a "disrupted gene" refers to a mutant gene that has a mutation that results in a premature stop codon. The disrupted gene product is truncated compared to the full-length, undisrupted gene product.

[0057] As used herein, the term "non-homologous end joining (NHEJ) pathway" refers to a pathway that repairs double-strand breaks in DNA by directly ligating the broken ends without the need for a homologous template. Template-independent religation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random microinsertions and microdeletions (indels) at the DNA breakpoint. This method can be used to intentionally disrupt, delete, or change the reading frame of targeted gene sequences. NHEJ generally uses short homologous DNA sequences, called microhomologies, to guide repair. These microhomologies are often present in single-stranded overhangs at the ends of double-strand breaks. When the overhangs are perfectly matched, NHEJ usually repairs the break accurately, while imprecise repairs resulting in polynucleotide loss can also occur, which is much more common when the overhangs are mismatched.

[0058] As used herein, a "normal gene" refers to a gene that has not undergone alteration, such as the loss, gain, or exchange of genetic material. A normal gene undergoes normal gene transmission and gene expression.

[0059] As used herein, "nuclease-mediated NHEJ" refers to NHEJ that is initiated after a nuclease, such as Cpf1 endonuclease, makes a double-stranded DNA break.

[0060] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two polynucleotides that are covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0061] Nucleic acids can be single-stranded or double-stranded, and can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, where the nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0062] As used herein, "operably linked" means that the expression of a gene is under the control of a promoter that is spatially linked to the gene. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene that the promoter controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0063] As used herein, "partially functional" describes a protein that is encoded by a mutated gene and that has less biological activity than a functional protein, but more biological activity than a non-functional protein.

[0064] As used interchangeably herein, a "premature stop codon" or "out-of-frame stop codon" refers to a nonsense mutation in the sequence of DNA that results in a stop codon at a position not normally found in the wild-type gene. A premature stop codon can result in a truncated or shortened protein compared to the full-length form of the protein.

[0065] "Promoter" as used herein means a synthetic or naturally derived molecule capable of conferring, activating or promoting expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further promote expression and / or to alter the spatial expression and / or temporal expression of the same. A promoter can also contain distal enhancer or repressor elements, which can be located as far away as several thousand base pairs from the start site of transcription. Promoters can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter can constitutively or variably regulate the expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, toxins, metal ions or inducers. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, the human U6 (hU6) promoter and the CMV IE promoter.

[0066] "Skeletal muscle" as used herein refers to a type of striated muscle that is under the control of the somatic nervous system and is attached to bones by bundles of collagen fibers known as tendons. Skeletal muscle is composed of individual components known as myocytes, or "muscle cells," sometimes colloquially called "muscle fibers." Muscle cells are formed from the fusion of developmental myoblasts (a type of embryonic precursor cell that gives rise to muscle cells) in a process known as myogenesis. These long, cylindrical, multinucleated cells are also called myofibers.

[0067] As used herein, "skeletal muscle condition" refers to conditions associated with skeletal muscle, such as muscular dystrophies, aging, muscle degeneration, wound healing, and muscle weakness or atrophy.

[0068] "Subject" and "patient", as used interchangeably herein, refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees), and humans). In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing other forms of therapy.

[0069] "Target gene" as used herein refers to any polynucleotide sequence that codes for a known or putative gene product. The target gene may be a mutated gene involved in a genetic disease. In certain embodiments, the target gene is the human dystrophin gene or the human B-cell lymphoma / leukemia 11A gene. In certain embodiments, the target gene is a mutated human dystrophin gene.

[0070] "Target region," as used herein, refers to the region of a target gene that a CRISPR / Cpf1-based gene editing system is designed to bind and cleave.

[0071] As used herein, "transgene" refers to a gene or genetic material that contains a gene sequence that is isolated from one organism and introduced into another organism. This non-native segment of DNA can retain the ability to produce RNA or protein in the transgenic organism, or it can change the normal function of the genetic code of the transgenic organism. The introduction of a transgene has the potential to change the phenotype of the organism.

[0072] "Variant," as used herein with respect to a nucleic acid, means (i) a portion or fragment of a reference polynucleotide sequence; (ii) the complement of a reference polynucleotide sequence, or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid, or its complement; or (iv) a nucleic acid that hybridizes to a reference nucleic acid under stringent conditions, its complement, or a sequence substantially identical thereto.

[0073] A "variant" with respect to a peptide or polypeptide differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with another amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is recognized in the art as generally involving minor changes. These minor changes can be determined to some extent by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic index can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that will result in a protein that retains biological function. Consideration of the hydrophilicity of an amino acid in the context of a peptide allows for the calculation of the maximum local average hydrophilicity of the peptide. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with this finding, it will be appreciated that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0074] As used herein, a "vector" refers to a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. For example, the vector may encode a Cpf1 endonuclease and at least one Cpf1 gRNA (e.g., a Cpf1 gRNA comprising any one of the polynucleotide sequences of SEQ ID NOs: 36-119) or its complement.

[0075] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the same meaning as commonly understood by those skilled in the art. For example, all academic terms used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein, and these techniques, are well known and commonly used in the art. The meaning and scope of terms should be clear; however, in case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0076] 2. CRISPR system The genetic construct (e.g., vector) of the present disclosure encodes a CRISPR / Cpf1-based gene editing system specific for the dystrophin gene (e.g., human dystrophin gene). "Clustered regularly interspaced short palindromic repeats" and "CRISPR", as used interchangeably herein, refer to a locus that contains multiple short direct repeats found in approximately 40% of sequenced bacterial and 90% of sequenced archaeal genomes. The CRISPR system is a microbial nuclease system that is involved in defense against invading phages and plasmids, providing a form of adaptive immunity. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated between the CRISPR repeats of the genome and act as a "memory" of past exposure.

[0077] Three classes of CRISPR systems are known: type I, type II, and type III effector systems. Type II effector systems use a single effector enzyme, such as Cpf1 endonuclease, to perform target DNA double-strand breaks in four successive steps to cleave dsDNA. Compared to type I and type III effector systems, which require multiple different effectors acting as a complex, type II effector systems can function in alternative contexts, such as eukaryotic cells. If the correct PAM is also present at the 5' end of the protospacer, Cpf1 endonuclease mediates the cleavage of the target DNA.

[0078] The activity of the CRISPR / Cpf1 system has three stages: modification, formation of crRNA, and interference. In the modification stage, Cas1 and Cas2 proteins facilitate modification of small pieces of DNA into CRISPR sequences. During the formation of crRNA, processing of pre-cr-RNA occurs to produce mature crRNA that guides Cas protein, i.e., Cpf1 endonuclease. In the interference stage: Cpf1 binds to crRNA to form a binary complex and identifies and cleaves the target DNA sequence.

[0079] In this system, the Cpf1 endonuclease is directed to a genomic target site by a synthetically reconstituted Cpf1 "guide RNA" ("Cpf1 gRNA"). Cpf1 endonuclease makes one strand longer than the other, generating "sticky" ends, e.g., sticky ends 4-5 nucleotides long, unlike Cas9, which generates blunt ends. Cpf1 endonuclease also cleaves target DNA more distal from the PAM compared to Cas9.

[0080] The target gene (e.g., dystrophin gene, e.g., human dystrophin gene) may be involved in cell differentiation or any other process where gene activation may be desired, or may have a mutation (e.g., frameshift mutation or nonsense mutation). If the target gene has a mutation that creates a premature stop codon, an ectopic splice acceptor site, or an ectopic splice donor site, the CRISPR / Cpf1-based gene editing system can be designed to recognize and bind to the polynucleotide sequence upstream or downstream of the premature stop codon, the ectopic splice acceptor site, or the ectopic splice donor site. The CRISPR / Cpf1-based system can also be used to disrupt normal gene splicing by targeting splice acceptor and splice donor to induce skipping of premature stop codon, or by restoring the disrupted reading frame. The CRISPR / Cpf1-based gene editing system may or may not mediate off-target changes to the protein-coding region of the genome.

[0081] Provided herein is a CRISPR / Cpf1-based gene editing system for use in genome editing and the treatment of genetic disease. The unique capability of the CRISPR / Cpf1-based gene editing system is its straightforward capability to simultaneously target multiple different genomic loci by co-expressing a single Cpf1 endonuclease with two or more Cpf1 gRNAs. The CRISPR / Cpf1-based gene editing system can be designed to target any gene, including genes involved in genetic disease, aging, tissue regeneration, or wound healing. The CRISPR / Cpf1-based gene editing system can include a Cpf1 endonuclease and at least one Cpf1 gRNA. In certain embodiments, the system includes two Cpf1 gRNAs.

[0082] Cpf1 endonuclease The CRISPR / Cpf1-based gene editing system can include Cpf1 endonuclease, which is an endonuclease that cleaves nucleic acids. Whereas Cas9 generates a blunt end 3 nucleotides upstream of the PAM site, Cpf1 endonuclease cuts in a staggered manner, generating a 5-nucleotide 5' overhang 18-23 bases away from the PAM.Cpf1 endonuclease may be derived from any bacterial or archaeal species, including, but not limited to, Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens or Porphyromonas macacae. In certain embodiments, the Cpf1 endonuclease is a Cpf1 endonuclease derived from Lachnospiraceae bacterium ND2006 ("LbCpf1") or Acidaminococcus ("AsCpf1").

[0083] In some embodiments, the Cpf1 endonuclease may comprise the humanized AsCpf1 sequence shown below (SEQ ID NO: 124): [ka] [ka] [ka] [ka] [ka]

[0084] In some embodiments, the Cpf1 endonuclease may comprise the humanized LbCpf1 sequence shown below (SEQ ID NO: 125): [ka] [ka] [ka] [ka] [ka]

[0085] Cpf1 endonuclease can interact with one or more Cpf1 gRNAs and in association with Cpf1 gRNA localizes to a site that includes a target domain and, in certain embodiments, a PAM sequence. In certain embodiments, the ability of Cpf1 endonuclease to interact with and cleave a target nucleic acid depends on the PAM sequence. The PAM sequence is a sequence within the target nucleic acid. In certain embodiments, cleavage of the target nucleic acid occurs upstream of the PAM sequence. Cpf1 endonuclease from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In certain embodiments, Cpf1 endonuclease recognizes the PAM of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0086] In certain embodiments, the vector encodes at least one Cpf1 endonuclease that recognizes the PAMs TTTA (SEQ ID NO:120), TTTG (SEQ ID NO:121), TTTC (SEQ ID NO:122), or TTTT (SEQ ID NO:123). In certain embodiments, the at least one Cpf1 endonuclease is a Cpf1 endonuclease from Lachnospiraceae bacterium ("LbCpf1") or Acidaminococcus ("AsCpf1"). In certain embodiments, the Cpf1 endonuclease is encoded by the polynucleotide sequence of SEQ ID NO:124 or SEQ ID NO:125.

[0087] The nucleic acid encoding Cpf1 endonuclease may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule may be chemically modified. The synthetic nucleic acid sequence may be codon-optimized, for example, at least one uncommon or less common codon is replaced with a common codon. For example, the synthetic nucleic acid may direct the synthesis of an optimized messenger mRNA, for example, optimized for expression in a mammalian expression system as described herein.

[0088] Additionally or alternatively, the nucleic acid encoding the Cpf1 endonuclease may contain a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0089] b. Cpf1 gRNA The CRISPR / Cpf1-based gene editing system includes at least one Cpf1 gRNA, for example, one Cpf1 gRNA, two Cpf1 gRNAs, three gRNAs, etc. The gRNA achieves the targeting of the CRISPR / Cpf1-based gene editing system. The Cpf1 gRNA can target any desired DNA sequence by replacing the sequence encoding the protospacer that confers targeting specificity to the desired DNA target. "Target region", "target sequence" or "protospacer" as used interchangeably herein refers to the region of the target gene (e.g., dystrophin gene) that the CRISPR / Cpf1-based gene editing system targets. The target sequence or protospacer is preceded by a PAM sequence at the 5' end of the protospacer. In some embodiments, the PAM sequence may be TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0090] In some embodiments, the protospacer may be about 17 bp to about 23 bp. In some embodiments, the Cpf1 gRNA comprises a polynucleotide sequence corresponding to the protospacer or a fragment thereof. In some embodiments, the Cpf1 gRNA may comprise a protospacer of about 17 bp to about 23 bp. In some embodiments, the protospacer of about 17 bp to about 23 bp is contiguous.

[0091] In some embodiments, the target region may comprise any one of the polynucleotide sequences of SEQ ID NOs: 1-35, any one of the fragments of SEQ ID NOs: 1-35, or a complement thereof. In some embodiments, the Cpf1 gRNA may comprise any one of the polynucleotide sequences of SEQ ID NOs: 36-119, any one of the fragments of SEQ ID NOs: 36-119, or a complement thereof. In some embodiments, the fragment of any one of SEQ ID NOs: 36-119 is about 17 bp to about 23 bp in length. In some embodiments, about 17 bp to about 23 bp in the fragment is contiguous.

[0092] A CRISPR / Cpf1-based gene editing system can include at least one Cpf1 gRNA, which target different DNA sequences. These target DNA sequences may overlap. The number of Cpf1 gRNAs encoded by a genetic construct (e.g., an AAV vector) of the present disclosure can be at least 1 Cpf1 gRNA, at least 2 different Cpf1 gRNAs, at least 3 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs, at least 5 different Cpf1 gRNAs, at least 6 different Cpf1 gRNAs, at least 7 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs, at least 9 different Cpf1 gRNAs, at least 10 different Cpf1 gRNAs, at least 11 different Cpf1 gRNAs, at least 12 different Cpf1 gRNAs, at least 13 different Cpf1 gRNAs, at least 14 different Cpf1 gRNAs, at least 15 different Cpf1 gRNAs, at least 16 different Cpf1 gRNAs, at least 17 different Cpf1 gRNAs, at least 18 different Cpf1 gRNAs, at least 19 different Cpf1 gRNAs, at least 20 different Cpf1 gRNAs, at least 21 different Cpf1 gRNAs, at least 22 different Cpf1 gRNAs, at least 23 different Cpf1 gRNAs, at least 24 different Cpf1 gRNAs, at least 25 different Cpf1 gRNAs, at least 26 different Cpf1 gRNAs, at least 27 different Cpf1 gRNAs, at least 28 different Cpf1 gRNAs, at least 29 different Cpf1 gRNAs, at least 30 different Cpf1 gRNAs, at least 31 different Cpf1 gRNAs, at least 32 different Cpf1 gRNAs, at least 33 different Cpf1 gRNAs, at least 34 different Cpf1 gRNAs, at least 35 different Cpf1 gRNAs, gRNAs, at least 20 different Cpf1 gRNAs, at least 25 different Cpf1 gRNAs, at least 30 different Cpf1 gRNAs, at least 35 different Cpf1 gRNAs, at least 40 different Cpf1 gRNAs, at least 45 different Cpf1 gRNAs, or at least 50 different Cpf1 gRNAs. The number of Cpf1 gRNAs encoded by the vector of the present disclosure may be at least 1 Cpf1 gRNA to at least 50 different Cpf1 gRNAs, at least 1 Cpf1 gRNA to at least 45 different Cpf1 gRNAs, at least 1 Cpf1 gRNA to at least 40 different Cpf1 gRNAs, at least 1 Cpf1 gRNA to at least 35 different Cpf1 gRNAs, at least 1 Cpf1 gRNA to at least 30 different Cpf1 gRNAs, at least 1 Cpf1 gRNA to at least 25 different Cpf1 gRNAs, at least 1 Cpf1 gRNA to at least 30 ...gRNA ~ at least 20 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 16 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 12 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 8 different Cpf1 gRNAs, at least 1 Cpf1 gRNA ~ at least 4 different Cpf1 gRNAs, at least 4 Cpf1 gRNA ~ at least 50 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 45 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 40 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 35 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 30 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 25 different Cpf1 gRNAs, at least 4 different Cpf1 gRNA ~ at least 20 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 16 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 12 different Cpf1 gRNAs, at least 4 different Cpf1 gRNAs ~ at least 8 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 50 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 45 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 40 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 35 different Cpf1 gRNAs, 8 different Cpf1 gRNAs ~ at least 30 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs ~ at least 25 different Cpf1 gRNAs, 8 different Cpf1 gRNAs ~ at least 20 different Cpf1 gRNAs, at least 8 different Cpf1 gRNAs to at least 16 different Cpf1 gRNAs, or 8 different Cpf1 gRNAs to at least 12 different Cpf1The gene construct (e.g., AAV vector) may be a gRNA. In certain embodiments, the gene construct (e.g., AAV vector) encodes one Cpf1 gRNA (i.e., the first gRNA) and optionally a Cpf1 endonuclease. In certain embodiments, the first gene construct (e.g., the first AAV vector) encodes one Cpf1 gRNA (i.e., the first Cpf1 gRNA) and optionally a Cpf1 endonuclease, and the second gene construct (e.g., the second AAV vector) encodes one Cpf1 gRNA (i.e., the second Cpf1 gRNA) and optionally a Cpf1 endonuclease.

[0093] 3. CRISPR / Cpf1-based gene editing system for genome editing of the dystrophin gene Gene construct The present invention is directed to a gene construct for genome editing, genome modification or gene expression modification of dystrophin gene (e.g., human dystrophin gene).The gene construct comprises at least one Cpf1 gRNA that targets human dystrophin gene sequence, such as Cpf1 endonuclease compatible target.The disclosed gRNA can be included in a CRISPR / Cpf1-based gene editing system, such as a system that uses Cpf1 endonuclease to target a region in dystrophin gene, for example, the intron region, splice acceptor site, and / or exon region surrounding exon such as exon 51 in human dystrophin gene, to cause genome deletion of this region and restore the expression of functional dystrophin in cells derived from DMD patients.

[0094] DMD is a severe muscle wasting disease caused by genetic mutations to the dystrophin gene. Dystrophin is a rod-shaped cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix through the cell membrane. Dystrophin provides structural stability to the dystroglycan complex in the cell membrane. The dystrophin gene is 2.2 vega bases at locus Xp21. The primary transcript measures approximately 2,400 kb, and the mature mRNA is approximately 14 kb. 79 exons code for a protein that is more than 3500 amino acids. Normal skeletal muscle tissue contains only small amounts of dystrophin, but the abnormal lack of expression of this dystrophin leads to the development of severe and incurable symptoms. Some mutations in the dystrophin gene result in the production of defective dystrophin and a severe dystrophic phenotype in affected patients. Some mutations in the dystrophin gene result in a partially functional dystrophin protein and a very mild dystrophic phenotype in affected patients.

[0095] DMD is the result of inherited or spontaneous mutations that cause nonsense or frameshift mutations in the dystrophin gene. Naturally occurring mutations and their consequences are relatively well understood for DMD. Mutations are typically deletions or duplications of regions of the gene that cause the protein to be out of frame and therefore completely dysfunctional. Removal of a single exon can be applied in as many as 83% of patients with frame correction that restores a nearly functional protein. CPF1 can target dystrophin exons and can be used to knock out a single exon by targeting splice acceptors or delete gene regions to remove single or multiple exons.

[0096] It is known that in-frame deletions occurring in the exon 45-55 region (e.g., exon 51) contained in the rod domain can produce a highly functional dystrophin protein, and many carriers are asymptomatic or exhibit mild symptoms. Furthermore, targeting exons in this region of the dystrophin gene (e.g., targeting exons of the dystrophin gene such as exon 51) could theoretically treat more than 60% of patients. Efforts have been made to restore the disrupted dystrophin reading frame in DMD patients by skipping non-essential exons (e.g., skipping exon 51) during mRNA splicing to produce an internally deleted but functional dystrophin protein. Deletion of an internal dystrophin exon (e.g., deletion of exon 51) causes a relatively mild form of Becker muscular dystrophy (i.e., BMD) that maintains the proper reading frame. The Becker muscular dystrophy (i.e., BMD) genotype is similar to DMD in that there is a deletion in the dystrophin gene. However, the deletion keeps the reading frame intact. Therefore, an internally truncated but partially functional dystrophin protein is produced. BMD has a wide range of phenotypes, but in many cases, the phenotype is much milder than DMD when the deletion is between exons 45 and 55 of dystrophin. Therefore, changing the DMD genotype to a BMD genotype is a common strategy to correct dystrophin. There are many strategies to correct dystrophin, and many of these rely on restoring the endogenous dystrophin reading frame. This shifts the disease genotype from DMD to Becker muscular dystrophy. Many BMD patients have intragenic deletions that maintain the translational reading frame, resulting in a shorter but largely functional dystrophin protein.

[0097] In certain embodiments, modification of exon 51 (e.g., deletion or elimination of exon 51, e.g., by NHEJ) to restore reading frame improves phenotype DMD subjects, such as DMD subjects with deletion mutations. In certain embodiments, exon 51 of the dystrophin gene refers to the 51st exon of the dystrophin gene. Exon 51 is frequently adjacent to frame-breaking deletions in DMD patients and has been targeted in clinical trials of oligonucleotide-based exon skipping. Clinical trials of the exon 51 skipping compound eteplirsen reported significant functional benefit over 48 weeks with an average of 47% dystrophin-positive fibers compared to baseline. Mutations at exon 51 are ideally suited for permanent correction by NHEJ-based genome editing.

[0098] The vector of the present disclosure can create a deletion in a dystrophin gene (e.g., human dystrophin gene). In certain embodiments, the vector is configured to form two double-strand breaks (first and second double-strand breaks) in two introns (first and second introns) adjacent to the target position of the dystrophin gene, thereby deleting the segment containing the dystrophin target position in the dystrophin gene. The "dystrophin target position" can be a dystrophin exon target position or a dystrophin intraexon target position as described herein. Deletion of the dystrophin exon target position can optimize the dystrophin sequence of a patient suffering from Duchenne muscular dystrophy, for example, increasing the function or activity of the encoded dystrophin protein, or improving the disease condition of the subject. In certain embodiments, the elimination of the dystrophin exon target position restores the reading frame. The dystrophin exon target position can include one or more exons of the dystrophin gene. In certain embodiments, the dystrophin target location comprises exon 51 of the dystrophin gene (eg, the human dystrophin gene).

[0099] The genetic construct (e.g., vector) of the present disclosure can mediate highly efficient gene editing at exon 51 of the dystrophin gene (e.g., human dystrophin gene). The genetic construct (e.g., vector) of the present disclosure restores expression of dystrophin protein in cells derived from DMD patients. Exon 51 is frequently adjacent to frame-breaking deletions in DMD. Removal of exon 51 from dystrophin transcripts by exon skipping can be used to treat approximately 15% of all DMD patients. This class of dystrophin mutations is ideally suited for permanent correction by NHEJ-based genome editing and HDR. The genetic construct (e.g., vector) described herein has been developed for targeted modification of exon 51 in the human dystrophin gene. The genetic construct (e.g., vector) of the present disclosure is transfected into human DMD cells and mediates efficient genetic modification and gene conversion to correct the reading frame. Protein recovery is simultaneous with frame recovery and is detected in the majority of cells treated with the CRISPR / Cpf1-based gene editing system.

[0100] Single or multiple gRNAs can be designed to restore dystrophin reading frame by targeting mutation hotspot at exon 51, or by introducing small insertions and deletions within exons, or by excluding exon 51. After treatment with the vector of the present disclosure, dystrophin expression can be restored in muscle cells of Duchenne patients in vitro. Human dystrophin was detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Significantly, the unique multiple gene editing capability of CRISPR / Cpf1-based gene editing system allows efficient generation of large deletions in this mutation hotspot region, which can correct up to 62% of patient mutations by universal or patient-specific gene editing approach. In some embodiments, candidate gRNAs are evaluated and selected based on off-target activity, on-target activity measured by surveyor, and distance from exons.

[0101] The Cpf1 gRNA may target a region of the dystrophin gene (DMD). In certain embodiments, the Cpf1 gRNA may target at least one of an exon, an intron, a promoter region, an enhancer region, a splice acceptor site, and / or a transcribed region of the dystrophin gene. In some embodiments, the target region comprises at least one polynucleotide sequence of SEQ ID NOs: 1-28. In certain embodiments, the Cpf1 gRNA targets intron 50 of the human dystrophin gene. In certain embodiments, the Cpf1 gRNA targets intron 51 of the human dystrophin gene. In certain embodiments, the Cpf1 gRNA targets exon 51 of the human dystrophin gene. The Cpf1 gRNA may comprise a polynucleotide sequence of any one of SEQ ID NOs: 36-64, 71-119, a fragment of any one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

[0102] 4. CRISPR / Cpf1-based gene editing system for genome editing of the B-cell lymphoma / leukemia 11A (BCL11a) gene Sickle cell anemia (SCA) is caused by point mutations in the β-globin gene, and β-thalassemia is caused by other mutations that lead to loss of β-globin expression. BCL11a is a transcriptional repressor that represses embryonic and fetal globin genes. Complete loss of BCL11a is embryonic lethal; however, disrupting the erythroid-specific enhancer region of BCL11a can reduce the abundance of the transcriptional repressor and increase fetal globin levels to ameliorate the disease phenotype. Similarly, certain mutations to the γ-globin (HBG1 / 2) promoter result in transcriptional repression and loss of hereditary high fetal hemoglobin (HPFH). The larger the indel footprint produced by Cpf1, the more efficiently it can disrupt the enhancer region of BCL11a or the repressor region of HBG1 / 2. In some embodiments, Cpf1 gRNA is designed to disrupt the enhancer region of BCL11a, increase fetal globin levels, and ameliorate the SCA phenotype. In some embodiments, the enhancer region comprises at least one polynucleotide sequence of SEQ ID NOs: 29-35. In some embodiments, the Cpf1 gRNA comprises a polynucleotide sequence of any one of SEQ ID NOs: 65-70, a fragment of any one of SEQ ID NOs: 65-70, or a complement thereof.

[0103] 5. DNA targeting compositions The present invention also covers a DNA targeting composition comprising such a gene construct. The DNA targeting composition comprises at least one Cpf1 gRNA (for example, one Cpf1 gRNA, two Cpf1 gRNAs, three gRNAs, etc.) that targets dystrophin gene (for example, human dystrophin gene) as described above. The at least one Cpf1 gRNA can bind to and recognize the target region. The target region can be selected immediately upstream of a possible out-of-frame stop codon, so that the insertion or deletion during the repair process restores the dystrophin reading frame by frame conversion. The target region can also be a splice acceptor or splice donor site, so that the insertion or deletion during the repair process disrupts splicing by disrupting splice sites and excluding exons, and restores the dystrophin reading frame. The target region can also be an ectopic stop codon, such that an insertion or deletion during the repair process restores the dystrophin reading frame by removing or destroying this stop codon.

[0104] In certain embodiments, the DNA targeting composition of the present disclosure comprises a first Cpf1 gRNA and a second Cpf1 gRNA, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence set forth in SEQ ID NOs: 36-119, or a complement thereof. In some embodiments, the polynucleotide sequence comprises at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof. In some embodiments, the polynucleotide sequence comprises at least one of SEQ ID NOs: 65-70, or a complement thereof. In certain embodiments, the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence.

[0105] In certain embodiments, the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0106] In certain embodiments, the DNA targeting composition may further comprise at least one Cpf1 endonuclease that recognizes the PAM TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123). In some embodiments, the DNA targeting composition comprises a Cpf1 endonuclease encoded by the polynucleotide sequence set forth in SEQ ID NO: 124 or SEQ ID NO: 125. In certain embodiments, the vector is configured to form a first and a second double-stranded break in the first and the second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene.

[0107] The deletion efficiency of the vector of the present disclosure can be related to the deletion size (i.e., the size of the segment deleted by this vector).In certain embodiments, the length or size of the specific deletion is determined by the distance between the PAM sequences in the gene to be targeted (e.g., the dystrophin gene).In certain embodiments, the specific deletion of a segment of the dystrophin gene (defined in terms of the length of this segment and the sequence that this segment contains (e.g., exon 51)) is the result of making a cut adjacent to a specific PAM sequence in the target gene (e.g., the dystrophin gene).

[0108] In a specific embodiment, the deletion size is about 50 to about 2,000 base pairs (bp), for example, about 50 to about 1999 bp, about 50 to about 1900 bp, about 50 to about 1800 bp, about 50 to about 1700 bp, about 50 to about 1650 bp, about 50 to about 1600 bp, about 50 to about 1500 bp, about 50 to about 1400 bp, about 50 to about 1300 bp, about 50 to about 1200 bp, about 50 to about 1150 bp, about 50 to about 1100 bp, about 50 to about 1000 bp, about 50 to about 900 bp, about 50 to about 850 bp, about 50 to about 800 bp, about 50 to about 75 ...900 bp, about 50 to about 1000 bp, about 50 to about 1100 bp, about 50 to about 1200 bp, about 50 to about 1300 bp, about 50 to about 1400 bp, about 50 to about 1500 bp, about 50 to about 1600 bp, about 50 to about 1700 bp, about 50 to about 1800 bp, about 50 to about 1900 bp, about 50 to about 1999 bp, about 50 to about 1900 bp, about Approx. 700bp, Approx. 50~Approx. 600bp, Approx. 50~Approx. 500bp, Approx. 50~Approx. 400bp, Approx. 50~Approx. 350bp, Approx. About 1900bp, about 100 to about 1800bp, about 100 to about 1700bp, about 100 to about 1650bp, about 100 to about 1600bp, about 100 to about 1500bp, about 100 to about 1400bp, about 100 to about 1300bp, about 100 to about 1200bp, about 100 to about 1150bp, about 100 to about 1100bp, about 100 to about 1000bp, about 100 to about 900bp, about 100 to about 850bp, about 100 to about 800bp, about 100 to about 750bp, about 100 to about 700bp, about 100 to about 600bp, about 100 to about 1000bp, about 100 to about 400bp, about 100 to about 350bp, about 100 to about 300bp, about 100 to about 250bp, about 100 to about 200bp, about 100 to about 150bp, about 200 to about 1999bp, about 200 to about 1900bp, about 200 to about 1800bp, about 200 to about 1700bp, about 200 to about 1650bp, about 200 to about 1600bp, about 200 to about 1500bp, about 200 to about 1400bp, about 200 to about 1300bp, about 200 to about 1200bp, about 200 to about 1150bp, about 200 to about 1100bp, about 200 to about 1000bp, about 200 to about 900bp, about 200 to about 850 bp, about 200 to about 800bp, about 200 to about 750bp, about 200 to about 700bp, about 200 to about 600bp, about 200 to about 2000bp, about 200 to about 400bp, about 200 to about 350bp, about 200 to about 300bp, about 200 to about 250bp, about 300 to about 1999bp,about 300 to about 1900 bp, about 300 to about 1800 bp, about 300 to about 1700 bp, about 300 to about 1650 bp, about 300 to about 1600 bp, about 300 to about 1500 bp, about 300 to about 1400 bp, about 300 to about 1300 bp, about 300 to about 1200 bp, about 300 to about 1150 bp, about 300 to about 1100 bp, about 300 to about 1000 bp, about 300 to about 900 bp, about 300 to about 850 bp, about 300 to about 800 bp, about 300 to about 750 bp, about 300 to about 700 bp, about 300 to about 600 bp, about 300 to about 3000 bp, about 300 to about 400 bp, or about 300 to about 350 bp. In certain embodiments, the deletion size can be about 118 base pairs, about 233 base pairs, about 326 base pairs, about 766 base pairs, about 805 base pairs, or about 1611 base pairs.

[0109] 6. Compositions for Gene Editing in Muscle The present invention is directed to a genetic construct (e.g., vector) or composition thereof for genome editing of a target gene in a subject's skeletal muscle or cardiac muscle. The composition comprises a modified AAV vector and a polynucleotide sequence encoding a CRISPR / Cpf1-based gene editing system (e.g., Cpf1 gRNA and Cpf1 endonuclease). The composition delivers an active CRISPR / Cpf1-based gene editing system to skeletal muscle or cardiac muscle. The genetic construct (e.g., vector) of the present disclosure can be used in correcting or reducing the effects of mutations in the dystrophin gene involved in genetic disease and / or other skeletal or cardiac muscle conditions (e.g., DMD). The composition can further comprise donor DNA or a transgene. The composition can be used in genome editing, genome manipulation, and correcting or reducing the effects of mutations in genes involved in genetic disease and / or other skeletal or cardiac muscle conditions.

[0110] A. CRISPR / Cpf1-based gene editing system for targeting dystrophin Disclosed herein is a CRISPR / Cpf1-based gene editing system specific for the dystrophin gene. The CRISPR / Cpf1-based gene editing system can include Cpf1 endonuclease and at least one Cpf1 gRNA for targeting the dystrophin gene. The CRISPR / Cpf1-based gene editing system can bind to and recognize the target region. The target region can be selected immediately upstream of a possible out-of-frame stop codon, so that the insertion or deletion during the repair process restores the dystrophin reading frame by frame conversion. The target region can also be a splice acceptor or splice donor site, so that the insertion or deletion during the repair process disrupts splicing and restores the dystrophin reading frame by disrupting the splice site and excluding the exon. The target region can also be an ectopic stop codon, so that the insertion or deletion during the repair process restores the dystrophin reading frame by removing or destroying this stop codon.

[0111] The Cpf1 gRNA can target a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-35, or its complement. For example, the CRISPR / Cpf1-based gene editing system of the present disclosure was engineered to mediate more efficient gene editing at exon 51 of the dystrophin gene. The CRISPR / Cpf1-based gene editing system restored dystrophin protein expression in cells from DMD patients.

[0112] B adeno-associated virus vector The composition can also include a viral delivery system. In certain embodiments, the vector is an adeno-associated virus (AAV) vector. The AAV vector is a small virus belonging to the genus Dependovirus of the family Parvoviridae that infects humans and some other primate species. The AAV vector can be used to deliver CRISPR / Cpf1-based gene editing systems using various construct structures. For example, the AAV vector can deliver Cpf1 endonuclease and Cpf1 gRNA expression cassette on separate vectors or on the same vector. Alternatively, both Cpf1 endonuclease and up to two gRNA expression cassettes can be combined in a single AAV vector within the packaging limit of 4.7 kb.

[0113] In certain embodiments, the AAV vector is a modified AAV vector. The modified AAV vector can have enhanced tissue tropism of cardiac muscle and skeletal muscle. The modified AAV vector can allow the delivery and expression of CRISPR / Cpf1-based gene editing system in mammalian cells. For example, the modified AAV vector can be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector can deliver nuclease to skeletal muscle and cardiac muscle in vivo. The modified AAV vector can be based on one or more of several capsid types (e.g., AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9). The modified AAV vector can be based on AAV2 pseudotypes with alternative muscle-tropic AAV capsids, for example, AAV2 / 1, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2.5 and AAV / SASTG vectors that efficiently transduce skeletal or cardiac muscles by systemic and local delivery (Seto et al. Current Gene Therapy (2012) 12: 139-151). The modified AAV vector can be AAV2i8G9 (Shen et al. J. Biol. Chem. (2013) 288: 28814-28823).

[0114] 7. Methods for gene editing in muscle The present disclosure is directed to a method of gene editing in skeletal muscle or cardiac muscle of a subject. The method includes administering the above-described composition for gene editing in skeletal muscle or cardiac muscle to the skeletal muscle or cardiac muscle of a subject. The genome editing can include correcting a mutant gene or inserting a transgene. Correcting a mutant gene can include deleting, re-editing or replacing the mutant gene. Correcting a mutant allergen can include nuclease-mediated NHEJ or HDR.

[0115] 8. Method of Treating a Subject by Correcting a Mutant Gene The subject matter of the present disclosure provides a method for correcting a mutant gene (e.g., a mutant dystrophin gene, e.g., a mutant human dystrophin gene) in a cell and treating a subject suffering from a genetic disease (e.g., DMD). The method can include administering to a cell or a subject a genetic construct (e.g., a vector) of the present disclosure or a composition comprising the genetic construct described above for gene editing in skeletal muscle or in cardiac muscle of a subject. The use of a genetic construct (e.g., a vector) of the present disclosure or a composition comprising the genetic construct for gene editing in skeletal muscle or cardiac muscle of a subject to deliver a CRISPR / Cpf1-based gene editing system to skeletal muscle or cardiac muscle can restore the expression of a fully functional or partially functional protein together with a repair template or donor DNA (which can replace the entire gene or the region containing the mutation). The CRISPR / Cpf1-based gene editing system can be used to introduce site-specific double-strand breaks at targeted genomic loci. Site-specific double-strand breaks are generated when the CRISPR / Cpf1-based gene editing system binds to the target DNA sequence, allowing cleavage of the target DNA. This DNA cleavage can stimulate natural DNA repair mechanisms, resulting in one of two possible repair pathways: the homology-directed repair (HDR) pathway or the non-homologous end joining (NHEJ) pathway.

[0116] The present disclosure is directed to gene editing by a CRISPR / Cpf1-based gene editing system without a repair template, which can efficiently correct the reading frame and restore the expression of functional proteins involved in genetic diseases. The CRISPR / Cpf1-based gene editing system of the present disclosure can include the use of homology-directed repair or nuclease-mediated non-homologous end joining (NHEJ)-based correction methods, which allow efficient correction in growth-limited primary cell lines that may not be amenable to homology-directed or selection-based gene correction. This strategy integrates the rapid and robust assembly of an active CRISPR / Cpf1-based gene editing system with an efficient gene editing method for treating genetic diseases caused by mutations in non-essential coding regions that generate frameshifts, premature stop codons, ectopic splice donor sites, or ectopic splice acceptor sites.

[0117] A nuclease-mediated nonhomologous end joining Restoration of protein expression from endogenous mutant gene can be by template-free NHEJ-mediated DNA repair. In contrast to the transient method of targeting target gene RNA, the correction of the target gene reading frame in genome by the transiently expressed CRISPR / Cpf1-based gene editing system can result in permanent restoration of target gene expression by each modified cell and all of its progeny. In certain embodiments, NHEJ is nuclease-mediated NHEJ, which in certain embodiments refers to NHEJ initiated by Cpf1 endonuclease to break double-stranded DNA. This method includes administering the gene construct (e.g., vector) of the present disclosure or a composition comprising this gene construct to skeletal muscle or cardiac muscle of a subject for gene editing in skeletal muscle or cardiac muscle.

[0118] Nuclease-mediated NHEJ gene correction corrects mutated target genes and can offer several potential advantages over HDR pathways. For example, NHEJ does not require a donor template, which can lead to non-specific insertion mutations. In contrast to HDR, NHEJ operates efficiently in all phases of the cell cycle, and can therefore be efficiently utilized in both cycling and post-mitotic cells (such as muscle fibers). This provides a robust and permanent gene repair alternative to oligonucleotide-based exon skipping or drug-imposed read-through of stop codons, and may theoretically require only one drug treatment. NHEJ-based gene correction using CRISPR / Cpf1-based gene editing systems and other genetically engineered nucleases, including meganucleases and zinc finger nucleases, can be combined with other existing ex vivo and in vivo platforms for cell and gene-based therapy, in addition to the plasmid electroporation approach described herein. For example, delivery of CRISPR / Cpf1-based gene editing systems by mRNA-based transfection or as purified cell-permeable proteins could enable a DNA-free genome editing approach that would avoid any possibility of insertional mutagenesis.

[0119] B homology-directed repair Restoring protein expression from endogenous mutated gene can include homologous recombination repair.The method as described above further includes administering a donor template to the cell.The donor template can include a polynucleotide sequence encoding a fully functional or partially functional protein.For example, the donor template can include a miniaturized dystrophin construct (called "minidys"), a fully functional dystrophin construct for repairing mutated dystrophin gene, or a fragment of the dystrophin gene that results in the repair of mutated dystrophin gene after homologous recombination repair.

[0120] C. Methods for correcting mutant genes and treating subjects using a CRISPR / Cpf1-based gene editing system The present disclosure also covers genome editing using CRISPR / Cpf1-based gene editing system to restore the expression of fully functional or partially functional proteins using repair template or donor DNA (which can replace the whole gene or the region containing the mutation). CRISPR / Cpf1-based gene editing system can be used to introduce site-specific double-strand breaks at targeted genomic loci. Site-specific double-strand breaks are created when CRISPR / Cpf1-based gene editing system uses gRNA to bind to target DNA sequence, thereby allowing target DNA to be cut. CRISPR / Cpf1-based gene editing system has the advantage of genome editing advancement due to its fast successful and efficient gene modification. This DNA break can stimulate natural DNA repair mechanism and lead to one of two possible repair pathways: homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathway. For example, a CRISPR / Cpf1-based gene editing system directed to the dystrophin gene can include a Cpf1 gRNA having a nucleic acid sequence of any one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

[0121] The present disclosure is directed to genome editing using a repair template-free CRISPR / Cpf1-based gene editing system that can efficiently correct reading frames and restore the expression of functional proteins involved in genetic diseases. The disclosed CRISPR / Cpf1-based gene editing system and method can include using homology-directed repair or nuclease-mediated non-homologous end joining (NHEJ)-based correction methods, which allow efficient correction in growth-limited primary cell lines where homology-directed or selection-based gene correction may not be applicable. This strategy integrates the rapid and robust assembly of an active CRISPR / Cpf1-based gene editing system with an efficient gene editing method for the treatment of genetic diseases caused by mutations in non-essential coding regions that result in frameshifts, premature stop codons, ectopic splice donor sites, or ectopic splice acceptor sites.

[0122] The present disclosure provides a method for correcting mutant genes in cells and treating subjects suffering from genetic diseases such as DMD. The method can include administering a CRISPR / Cpf1-based gene editing system, a polynucleotide or vector encoding the CRISPR / Cpf1-based gene editing system, or a composition of the CRISPR / Cpf1-based gene editing system to cells or subjects as described above. The method can include administering a CRISPR / Cpf1-based gene editing system, for example, administering a Cpf1 endonuclease, a polynucleotide sequence encoding the Cpf1 endonuclease, and / or at least one Cpf1 gRNA, where the gRNAs target different DNA sequences. These target DNA sequences can be overlapping. The number of gRNAs administered to the cells can be at least one gRNA, at least two different gRNAs, at least three different gRNAs, at least four different gRNAs, at least five different gRNAs, at least six different gRNAs, at least seven different gRNAs, at least eight different gRNAs, at least nine different gRNAs, at least ten different gRNAs, at least fifteen different gRNAs, at least twenty different gRNAs, at least thirty different gRNAs, or at least fifty different gRNAs, as described above. The gRNAs can comprise at least one nucleic acid sequence of SEQ ID NOs: 36-64, 71-119, or a complement thereof. The method can include homology directed repair or non-homologous end joining.

[0123] 9. Methods of Treating Disease The present disclosure is directed to a method of treating a subject in need thereof. The method comprises administering to a tissue of the subject a genetic construct (e.g., vector) of the present disclosure described above or a composition comprising the genetic construct. In certain embodiments, the method can comprise administering to a skeletal or cardiac muscle of the subject a genetic construct (e.g., vector) of the present disclosure described above or a composition comprising the genetic construct. In certain embodiments, the method can comprise administering to a vein of the subject a genetic construct (e.g., vector) of the present disclosure described above or a composition comprising the genetic construct. In certain embodiments, the subject suffers from a condition of the skeletal or cardiac muscle that causes degeneration or debilitation or a genetic disease. For example, the subject suffers from Duchenne muscular dystrophy described above.

[0124] A. Duchenne muscular dystrophy Using the above-described method, dystrophin gene can be corrected to restore the fully functional or partially functional protein expression of said mutant dystrophin gene.In some aspects and embodiments, the present disclosure provides a method for reducing the effect (e.g., clinical symptoms / indications) of DMD in a patient.In some aspects and embodiments, the present disclosure provides a method for treating DMD in a patient.In some aspects and embodiments, the present disclosure provides a method for preventing DMD in a patient.In some aspects and embodiments, the present disclosure provides a method for preventing further deterioration of DMD in a patient.

[0125] 10. Constructs and Plasmids The composition described above can include a genetic construct encoding the CRISPR / Cpf1-based gene editing system disclosed herein. The genetic construct (e.g., a plasmid) can include a nucleic acid encoding the CRISPR / Cpf1-based gene editing system (e.g., at least one of Cpf1 endonuclease and / or Cpf1 gRNA). The composition described above can include a genetic construct encoding a modified AAV vector and a nucleic acid sequence encoding the CRISPR / Cpf1-based gene editing system disclosed herein. The genetic construct (e.g., a plasmid) can include a nucleic acid encoding the CRISPR / Cpf1-based gene editing system. The composition described above can include a genetic construct encoding a modified lentiviral vector disclosed herein.

[0126] In some embodiments, the genetic construct may comprise a promoter operably linked to a polynucleotide sequence encoding at least one Cpf1 gRNA and / or Cpf1 endonuclease. In some embodiments, the promoter is operably linked to a polynucleotide sequence encoding a first Cpf1 gRNA, a second Cpf1 gRNA, and / or a Cpf1 endonuclease. The genetic construct can be present in a cell as a functional extrachromosomal molecule. The genetic construct can be a linear minichromosome, such as a centromere, a telomere, or a plasmid or cosmid.

[0127] Gene constructs can also be part of the genome of recombinant virus vectors, including recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses. Gene constructs can be part of the genetic material in attenuated live microorganisms or recombinant microbial vectors that live in cells. Gene constructs can include regulatory elements for gene expression of the coding sequence of nucleic acid. Regulatory elements can be promoters, enhancers, start codons, stop codons, or polyadenylation signals.

[0128] In a particular embodiment, the genetic construct is a vector. The vector may be an adeno-associated virus (AAV) vector, which encodes at least one Cpf1 endonuclease and at least one Cpf1 gRNA; the vector can express at least one Cpf1 endonuclease and at least one Cpf1 gRNA in mammalian cells. The vector may be a plasmid. The vector can be used for in vivo gene therapy. The vector may be recombinant. The vector may include a heterologous nucleic acid encoding a CRISPR / Cpf1-based gene editing system. The vector may be a plasmid. The vector may be useful for transfecting a nucleic acid encoding a CRISPR / Cpf1-based gene editing system into a cell, and the transformed host cell is maintained in culture under conditions that allow expression of the CRISPR / Cpf1-based gene editing system.

[0129] Coding sequences can be optimized for stability and high levels of expression. In some cases, codons are selected to reduce secondary RNA structure formation, such as those formed due to intramolecular binding.

[0130] The vector can include a heterologous nucleic acid encoding a CRISPR / Cpf1-based gene editing system, and can further include a start codon that can be upstream of the CRISPR / Cpf1-based gene editing system coding sequence, and a stop codon that can be downstream of the CRISPR / Cpf1-based gene editing system coding sequence. The start and stop codons can be in frame with the CRISPR / Cpf1-based gene editing system coding sequence. The vector can also include a promoter operably linked to the CRISPR / Cpf1-based gene editing system coding sequence. The promoter operably linked to the CRISPR / Cpf1-based gene editing system coding sequence can be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, a U6 promoter, such as a human U6 promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a tissue-specific promoter, such as a muscle- or skin-specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Publication Nos. 20040175727 and 20040192593, the contents of which are incorporated herein in their entireties.Examples of muscle-specific promoters include the Spc5-12 promoter (described in U.S. Patent Publication No. 20040192593, which is incorporated by reference in its entirety; Hakim et al. Mol. Ther. Methods Clin. Dev. (2014) 1:14002; and Lai et al. Hum Mol Genet. (2014) 23(12):3189-3199), the MHCK7 promoter (described in Salva et al., Mol. Ther. (2007) 15:320-329), the CK8 promoter (described in Park et al. PLoS ONE (2015) 10(4):e0124914), and the CK8e promoter (described in Muir et al., Mol. Ther. Methods Clin. Dev. (2014) 1:14025). In some embodiments, expression of the gRNA and / or Cpf1 endonuclease is driven by tRNA.

[0131] Each of the polynucleotide sequences encoding Cpf1 gRNA and / or Cpf1 endonuclease can be operably linked to a promoter. The promoters operably linked to Cpf1 gRNA and / or Cpf1 endonuclease can be the same promoter. The promoters operably linked to Cpf1 gRNA and / or Cpf1 endonuclease can be different promoters. The promoters can be constitutive, inducible, repressible or regulatable promoters.

[0132] The vector can also include a polyadenylation signal that can be downstream of the gene editing system based on CRISPR / Cpf1.The polyadenylation signal can be SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human β-globin polyadenylation signal.The SV40 polyadenylation signal can be the polyadenylation signal from pCEP4 vector (Invitrogen, San Diego, CA).

[0133] The vector can also include a CRISPR / Cpf1-based gene editing system, i.e., an enhancer upstream of the Cpf1 endonuclease coding sequence, the Cpf1 gRNA, or a CRISPR / Cpf1-based gene editing system. The enhancer can be essential for DNA expression. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as those derived from CMV, HA, RSV, or EBV. Polynucleotide function enhancers are described in U.S. Pat. No. 5,593,972, U.S. Pat. No. 5,962,428, and WO 94 / 016737, the contents of each of which are fully incorporated by reference. The vector can also include a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector in the cell. The vector can also include a regulatory sequence, which can be fully adapted for gene expression in the mammalian or human cell to which the vector is administered. Vectors may also include a reporter gene, such as green fluorescent protein ("GFP"), and / or a selectable marker, such as hygromycin ("Hygro").

[0134] The vector can be an expression vector or a system for producing the protein by conventional techniques and readily available starting materials (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), fully incorporated by reference). In some embodiments, the vector can include a nucleic acid sequence encoding a CRISPR / Cpf1-based gene editing system, for example, a nucleic acid sequence encoding a Cpf1 endonuclease and at least one Cpf1 gRNA comprising at least one nucleic acid sequence of SEQ ID NOs: 36-119 or a complement thereof.

[0135] 11. Pharmaceutical Compositions The subject of the present disclosure provides a composition comprising the genetic construct described above. The pharmaceutical composition of the present invention can be formulated according to the mode of administration used. When the pharmaceutical composition is an injectable pharmaceutical composition, the pharmaceutical composition is sterile, pyrogen-free and particulate-free. An isotonic formulation is preferably used. In general, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, an isotonic solution (e.g., phosphate buffered saline) is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0136] The composition can further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a functional molecule as a vehicle, adjuvant, carrier, or diluent. The pharmaceutically acceptable excipient can be a gene transfer promoter (which can include surfactants), such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known gene transfer promoters.

[0137] The gene transfer promoter is a polyanion, a polycation (including poly-L-glutamic acid (LGS)), or a lipid. The gene transfer promoter is poly-L-glutamic acid, and more preferably, poly-L-glutamic acid is present in a concentration of less than 6 mg / ml in the composition for genome editing in skeletal muscle or cardiac muscle. The gene transfer promoter can also include surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs (including monophosphoryl lipid A), muramyl peptides, quinone analogs, and vesicles, such as squalene and squalene, and hyaluronic acid can also be used with the gene construct. In some embodiments, the DNA vector encoding the composition may also contain a gene transfer facilitating agent, such as a lipid, a liposome (including lecithin liposomes or other liposomes known in the art), a DNA-liposome mixture (see, e.g., WO 09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known gene transfer facilitating agents. Preferably, the gene transfer facilitating agent is a polyanion, polycation (including poly-L-glutamic acid (LGS)), or a lipid.

[0138] 12. Method of Service Provided herein is a method of delivering the disclosed genetic construct (e.g., vector) or composition thereof to a cell. The delivery of the composition can be transfection or electroporation of the composition as a nucleic acid molecule that is expressed in the cell and delivered to the surface of the cell. The nucleic acid molecule can be electroporated using a BioRad Gene Pulser Xcell device or an Amaxa Nucleofector IIb device. Several different buffers can be used, such as BioRad electroporation solution, Sigma phosphate buffered saline product number D8537 (PBS), Invitrogen OptiMEM I (OM) or Amaxa Nucleofector solution V (NV). Transfection can include transfection reagents such as Lipofectamine 2000.

[0139] When the genetic construct or composition of the present disclosure is delivered to tissue, and the vector is delivered into the cells of a mammal, the transgenic cells express Cpf1 gRNA and Cpf1 endonuclease.The genetic construct or composition can be administered to a mammal to change gene expression, or re-edit or change genome.For example, the genetic construct or composition can be administered to a mammal to correct dystrophin gene in the mammal.The mammal can be human, non-human primate, cow, pig, sheep, goat, antelope, bison, buffalo, bovid, deer, hedgehog, elephant, llama, alpaca, mouse, rat or chicken, and preferably human, cow, pig or chicken.

[0140] The gene construct (e.g., vector) encoding Cpf1 gRNA and Cpf1 endonuclease can be delivered to mammals by DNA injection (also called DNA vaccination) with or without in vivo electroporation, liposome-mediated, nanoparticle-facilitated, and / or recombinant vector.The recombinant vector can be delivered by any virus type.The virus type can be recombinant lentivirus, recombinant adenovirus, and / or recombinant adeno-associated virus.

[0141] The genetic construct (e.g., vector) of the present disclosure or a composition comprising the genetic construct can be introduced into a cell to genetically correct the dystrophin gene (e.g., human dystrophin gene). In certain embodiments, the genetic construct (e.g., vector) of the present disclosure or a composition comprising the genetic construct is introduced into myoblasts derived from a DMD patient. In certain embodiments, the genetic construct (e.g., vector) or a composition comprising the genetic construct can be introduced into fibroblasts derived from a DMD patient, the genetically corrected fibroblasts can be treated with MyoD to induce differentiation into myoblasts, and the myoblasts can be transplanted into a subject (e.g., a damaged muscle of a subject) to verify that the corrected dystrophin protein is functional and / or to treat the subject. The modified cells can be stem cells (e.g., induced pluripotent stem cells), bone marrow-derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts derived from a DMD patient, CD133 + The cells can be mesoangioblasts, MyoD-transduced cells or Pax7-transduced cells, or other myogenic progenitor cells. For example, the CRISPR / Cpf1-based gene editing system can induce neuronal or myogenic differentiation of induced pluripotent stem cells.

[0142] 13. Route of Administration The genetic construct (e.g., vector) of the present disclosure or a composition comprising the genetic construct can be administered to a subject by various routes (e.g., orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof). In certain embodiments, the genetic construct (e.g., vector) or composition of the present disclosure is administered intramuscularly, intravenously, or a combination thereof to a subject (e.g., a subject suffering from DMD). For veterinary use, the genetic construct (e.g., vector) or composition of the present disclosure can be administered in an appropriately tolerated formulation according to normal veterinary practice. A veterinarian can readily determine the dosage regimen and route of administration that is most suitable for a particular animal. The composition can be administered by a conventional syringe, a needleless injection device, a "microprojectile bombardment gone gun," or other physical methods (e.g., electroporation ("EP"), "hydrodynamic methods," or ultrasound).

[0143] The genetic constructs (e.g., vectors) or compositions of the present disclosure can be delivered to mammals by several techniques (e.g., DNA injection with and without in vivo electroporation (also referred to as DNA vaccination), liposome-mediated, nanoparticle-facilitated, recombinant vectors (e.g., recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses)). The compositions can be injected into skeletal or cardiac muscles. For example, the compositions can be injected into the tibialis anterior muscle or tail.

[0144] In some embodiments, a genetic construct (e.g., a vector) of the present disclosure or a composition comprising this genetic construct is administered: 1) by tail vein injection (systemic) into adult mice; 2) by intramuscular injection, e.g., by local injection into a muscle (e.g., the TA or gastrocnemius) in adult mice; 3) by intraperitoneal injection into P2 mice; or 4) by facial vein injection (systemic) into P2 mice.

[0145] 14.Cell type Any of these delivery methods and / or routes of delivery can be utilized with a myriad of cell types (e.g., cell types currently under investigation for cell-based therapies for DMD), including, but not limited to, immortalized myoblasts, such as wild type and DMD patient derived lines, e.g., Δ48-50 DMD, DMD6594(del48-50), DMD8036(del48-50), C25C14 and DMD-7796 cell lines, primary DMD skin fibroblasts, induced pluripotent stem cells, bone marrow derived progenitor cells, skeletal muscle progenitor cells, human skeletal myoblasts from DMD patients, CD133 + Cells, mesodermal hemangioblasts, cardiomyocytes, hepatocytes, chondrocytes, mesenchymal progenitor cells, hematopoietic stem cells, smooth muscle cells, and MyoD- or Pax7-transduced cells, or other myogenic progenitor cells. Immortalization of human myogenic cells can be used to derive clones of genetically corrected myogenic cells. Cells can be modified ex vivo to isolate and grow clonal populations of immortalized DMD myoblasts that contain genetically corrected dystrophin genes and no other nuclease-induced mutations in the protein-coding region of the genome. Alternatively, transient delivery of CRISPR / Cpf1-based systems in vivo by non-viral or non-integrating viral transduction or by direct delivery of purified proteins and gRNAs containing cell-penetrating motifs can allow highly specific correction in situ with minimal or no risk of exogenous DNA integration.

[0146] 15. Kit Provided herein is a kit that can be used to correct mutant dystrophin gene.The kit includes at least Cpf1 gRNA for correcting mutant dystrophin gene and instructions for using CRISPR / Cpf1-based gene editing system.Also provided herein is a kit that can be used for genome editing of dystrophin gene in skeletal muscle or cardiac muscle.The kit includes the gene construct (e.g., vector) for genome editing in skeletal muscle or cardiac muscle described above or a composition comprising this gene construct, and instructions for using said composition.

[0147] The instructions included in the kit can be affixed to the packaging material or can be included as a package insert. The instructions are typically, but not limited to, written or printed material. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include an address of an internet site that provides the instructions.

[0148] The gene construct (for example, vector) for correcting mutant dystrophin in skeletal muscle or cardiac muscle or for genome editing of dystrophin gene or the composition comprising this gene construct can comprise the modified vector comprising the Cpf1 gRNA and Cpf1 endonuclease described above, which specifically binds to and cuts a certain region of dystrophin gene.The gene editing system based on CRISPR / Cpf1 described above can be included in the kit to specifically bind to and target a specific region in mutant dystrophin gene.The kit can further comprise donor DNA, another gRNA or transgene described above.

[0149] The kit may further optionally include one or more components of reagents required for use of the disclosed compositions or for facilitating quality control evaluation, such as standards, buffers, diluents, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc. Other components such as buffers and solutions for cell isolation and / or processing can be included in the kit. The kit can further include one or more controls. One or more components of the kit can be lyophilized, in which case the kit can further include reagents suitable for reconstituting the lyophilized components. EXAMPLES

[0150] 16. Working Example It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having thus described the present disclosure in detail, the disclosure will be more clearly understood by reference to the following examples. The examples are merely intended to illustrate some aspects and embodiments of the present disclosure, and should not be considered as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents and publications referred to herein are incorporated herein by reference in their entirety.

[0151] The present invention has multiple aspects and is illustrated by the following non-limiting examples.

[0152] Example 1 Guide RNA design and material preparation Cpf1 from Acidaminococcus was obtained from the Addgene non-profit plasmid depository (pY010(pcDNA3.1-hAsCpf1; “AsCPF1 plasmid”) from Feng Zhang (Addgene plasmid #69982)). The AsCPF1 plasmid was transformed into chemically competent E. coli, amplified, and sequenced. Cpf1 guide RNA (also known as Cpf1 crRNA) was designed to target splice sites on the predominant exon mutations in dystrophin and enhancers of BCL11a using the University of California Santa Cruz Genome Browser program, ordered as oligomers from Integrated DNA Technologies (IDT), prepared by PCR, and column purified as previously described (Zetsche et al., Cell 163(3):759-71(2015)).

[0153] Guide RNA validation. Transfection of HEK293 cells (ATCC) was performed using Lipofectamine 2000 in 24-well plates according to the manufacturer's recommendations. Each well received 400 ng of AsCPF1 plasmid and 100 ng of U6::sgRNA PCR product. After 72 h, cells were isolated and genomic DNA was purified with DNeasy columns (QIAGEN). Surveyor nuclease digestion (IDT) and deletion PCR were performed using primers flanking the genomic region of interest as previously described (Ousterout et al., Nature Communications 6:6244 (2015); Guschin et al., Methods Mol. Biol. 649:247-256 (2010)). Digested PCR products were electrophoresed in TBE gels (Invitrogen) for 30 min at 200 V, stained with ethidium bromide (EtBr) and imaged on Gel Doc™ (Biorad). Deletion PCR products were electrophoresed in 1% agarose gels for 30 min at 120 V, stained with EtBr and imaged on Gel Doc™ (Biorad).

[0154] Example 2 Dystrophin splice receptor guide RNA We designed 15 guide RNAs targeting the top highly mutated dystrophin exons by targeting the cleavage region as close as possible to the splice acceptor, which was achieved by the presence of available PAMs (Table 1). When possible, multiple guide RNAs were targeted to the same splice acceptor. Candidate guide RNAs were screened in vitro. Guide RNAs that showed immediate positive results included those targeting exon 44, exon 46, and exon 51 (Figure 2A-2C). Surveyor nuclease digestion was detected in guide RNAs targeting the exon 44 splice acceptor (Figure 2A), the exon 46 splice acceptor (Figure 2B), and the 3' end of exon 51 (Figure 2C). Figure 2D revealed that guide RNAs targeting the exon 51 splice acceptor and the 3' end of exon 51 could be used to cause gene deletion, indicating the activity of guide RNAs targeting exon 51.

[0155] Table 1 shows the design of guide RNAs targeting dystrophin exons. The PAM sequence (TTTN) is underlined. The sense guide RNA has TTTN at the 5' end. The antisense guide RNA has NAAA PAM at the 3' end.

[0156] [Table 1]

[0157] Example 3 Compatible overhang deletion of exon 51 To determine whether guide RNAs with compatible overhang sequences would promote seamless deletions, six guide RNAs were designed in intron 50 and seven guide RNAs were designed in intron 51 (Table 2) to generate compatible overhang deletions. 42 unique gRNA pairs (6x7) were tested and screened for deletion activity, i.e., targeting exon 51 deletions. The set included three overhang-compatible pairs (see Table 2). Seven pairs were confirmed for activity. Figure 3 is a representative image showing a smaller band, indicating the deletion of exon 51. These results demonstrate the first Cpf1-targeted splice acceptor disruption and the deletion of exon 51 in the dystrophin gene.

[0158] [Table 2]

[0159] Example 4 Targeted deletion of exon 51 in patient-derived myoblasts Patient-derived myoblasts with a deletion of exons 48–50 (Δ48–50) were cultured in skeletal muscle expansion medium. Electroporation was performed according to standard laboratory procedures. Cells were cultured for 3 days and evaluated for protein expression in patient-derived myoblasts (Figure 4) and genomic deletions caused by SaCas9 (Cas9 from Staphylococcus aureus) or LbCpf1 (CPF1 from Lachnospiraceae bacterium ND2006) (Figure 5). Figure 4 demonstrates that Western blots of HA-tagged SaCas9 and LbCpf1 show expression in extracted proteins 72 hours after plasmid transfection. Figure 5 demonstrates that PCR across the targeted genomic region shows smaller bands in bulk treated myoblasts with SaCas9 gRNA or Cpf1 crRNA, consistent with removal of exon 51 and the surrounding intronic portion.

[0160] Next, myoblasts were differentiated and then assessed for dystrophin transcript expression and exon 51 deletion (Figure 6). Figure 6 demonstrates that differentiated myoblasts expressed dystrophin transcripts lacking exon 51, as shown by a small band generated by RT-PCR, thus indicating that SaCas9 or LbCpf1 targeting exon 51 removed the exon 51 exon from the transcript.

[0161] A large panel of Cpf1 crRNAs was evaluated in HEK293 cells (Figure 7; see Table 3 for Cpf1 crRNA sequence). All Cpf1 crRNAs targeting exon 51 or surrounding introns used are listed in Table 3. As shown in Figure 7, HEK293 cells treated with a panel of crRNAs for 3 days showed variable activity by Surveyor® nuclease assay. Cpf1 crRNAs #38, 41, 42, 43, 45, 46, 47, 49, 54, 55, 56, 59, 63, 64, and 65 showed the highest activity as indicated by short bands.

[0162] [Table 3] TIFF0007633699000014.tif123153

[0163] Example 5 BCL11a enhancer targeting Potential candidates for increasing fetal globin levels in Sickle Cell Anemia (SCA) were designed. Guide RNAs of Cpf1 were designed to target the BCL11a enhancer region (Table 3) to generate potential candidates for increasing fetal globin levels in Sickle Cell Anemia (SCA). These reagents were designed to disrupt the BCL11a enhancer. These reagents will be tested in SCA cell models.

[0164] [Table 4]

[0165] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and should not be taken as limitations on the scope of the invention, which is defined solely by the appended claims and equivalents thereof.

[0166] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to, with respect to the chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof.

[0167] For reasons of completeness, the various aspects of the invention are presented in the following numbered clauses.

[0168] Section 1. A Cpf1 guide RNA (gRNA) targeting the dystrophin gene and comprising a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof.

[0169] Section 2. A DNA targeting composition comprising a Cpf1 endonuclease and at least one Cpf1 gRNA described in section 1.

[0170] Section 3. A DNA targeting composition comprising a first Cpf1 gRNA and a second Cpf1 gRNA, wherein the first Cpf1 gRNA and the second Cpf1 gRNA each comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and the first Cpf1 gRNA and the second Cpf1 gRNA target the dystrophin gene.

[0171] Clause 4. The DNA targeting composition of clause 3, wherein the first Cpf1 gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56, and the second Cpf1 gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:61.

[0172] Clause 5. The DNA targeting composition of clause 3 or clause 4, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0173] Item 6. A DNA targeting composition described in any one of items 3 to 5, further comprising Cpf1 endonuclease.

[0174] Clause 7. The DNA targeting composition of clause 2 or clause 6, wherein the Cpf1 endonuclease recognizes the protospacer adjacent motif (PAM) TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0175] Item 8. The Cpf1 endonuclease is capable of selectively isoforms of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus 8. The DNA targeting composition of claim 7, which is derived from a bacterial species selected from the group consisting of: Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae.

[0176] Item 9. The DNA targeting composition described in any one of items 6 to 8, wherein the Cpf1 endonuclease is derived from Lachnospiraceae bacterium ND2006 (LbCpf1) or Acidaminococcus (AsCpf1).

[0177] Item 10. The DNA targeting composition described in any one of items 6 to 9, wherein the Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO: 124 or SEQ ID NO: 125.

[0178] Item 11. An isolated polynucleotide comprising a polynucleotide sequence encoding the Cpf1 gRNA described in item 1 or the DNA targeting composition described in any one of items 2 to 10.

[0179] Paragraph 12. A vector comprising a polynucleotide sequence encoding the Cpf1 gRNA described in paragraph 1, a DNA targeting composition described in any one of paragraphs 2 to 10, or an isolated polynucleotide described in paragraph 10.

[0180] Clause 13. The vector of clause 12, further comprising a polynucleotide sequence encoding a Cpf1 endonuclease.

[0181] Section 14. A vector encoding (a) a first Cpf1 guide RNA (gRNA), (b) a second Cpf1 gRNA, and (c) at least one Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 gRNA comprise a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences.

[0182] Item 15. The vector of item 14, wherein the vector is configured to form first and second duplexes in first and second introns flanking exon 51 of the human DMD gene.

[0183] Clause 16. The vector of clause 14 or clause 15, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0184] Item 17. The vector according to any one of items 12 to 16, wherein the vector is a viral vector.

[0185] Item 18. The vector described in item 17, wherein the vector is an adeno-associated virus (AAV) vector.

[0186] Item 19. The vector described in any one of items 12 to 18, wherein the vector comprises a tissue-specific promoter operably linked to a polynucleotide sequence encoding the first Cpf1 gRNA, the second Cpf1 gRNA, and / or the Cpf1 endonuclease.

[0187] Item 20. The vector of item 19, wherein the tissue-specific promoter is a muscle-specific promoter.

[0188] Paragraph 21. A cell comprising a polynucleotide sequence encoding the Cpf1 gRNA described in paragraph 1, a DNA targeting composition described in any one of paragraphs 2 to 10, an isolated polynucleotide described in paragraph 11, or a vector described in any one of paragraphs 12 to 20.

[0189] Paragraph 22. A kit comprising a polynucleotide sequence encoding the Cpf1 gRNA described in paragraph 1, a DNA targeting composition described in any one of paragraphs 2 to 10, an isolated polynucleotide described in paragraph 11, a vector described in any one of paragraphs 12 to 20, or a cell described in paragraph 21.

[0190] 23. A composition for deleting a segment including exon 51 in a dystrophin gene, the composition comprising: (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a polynucleotide sequence encoding a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 The composition, wherein the gRNA comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, the first Cpf1 gRNA and the second Cpf1 gRNA comprise different polynucleotide sequences, and the first vector and the second vector are configured to form first and second double-stranded breaks in a first intron and a second intron, respectively, adjacent to exon 51 of the human DMD gene, thereby deleting a segment including exon 51 in the dystrophin gene.

[0191] Clause 24. The composition of clause 23, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0192] Item 25. The composition of item 23 or 24, wherein the first Cpf1 endonuclease and the second Cpf1 endonuclease are the same.

[0193] Clause 26. The composition of clause 23 or clause 24, wherein the first Cpf1 endonuclease and the second Cpf1 endonuclease are different.

[0194] Item 27. The composition of item 25 or 26, wherein the first Cpf1 endonuclease and / or the second Cpf1 endonuclease is a CPF1 endonuclease from Lachnospiraceae bacterium ND2006 (LbCpf1) and / or Acidaminococcus (AsCpf1).

[0195] Clause 28. The composition of any one of clauses 25 to 27, wherein the first Cpf1 endonuclease and / or the second Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO: 124 or SEQ ID NO: 125.

[0196] Item 29. The composition according to any one of items 23 to 28, wherein the first vector and / or the second vector is a viral vector.

[0197] Clause 30. The composition described in clause 29, wherein the first vector and / or the second vector is an adeno-associated virus (AAV) vector.

[0198] Item 31. The composition described in item 30, wherein the AAV vector is an AAV8 vector or an AAV9 vector.

[0199] Item 32. The composition according to any one of items 23 to 31, wherein the dystrophin gene is a human dystrophin gene.

[0200] Item 33. A composition according to any one of items 23 to 32 for use in medicine.

[0201] Item 34. A composition described in any one of items 23 to 32 for use in the treatment of Duchenne muscular dystrophy.

[0202] Item 35. A cell comprising the composition described in any one of items 23 to 34.

[0203] Clause 36. A modified adeno-associated viral vector for genome editing of a mutant dystrophin gene in a subject, comprising a first polynucleotide sequence encoding the Cpf1 gRNA described in clause 1, and a second polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

[0204] Clause 37. A method of correcting a mutant dystrophin gene in a cell, the method comprising administering to the cell a polynucleotide sequence encoding the Cpf1 gRNA described in clause 1, a DNA targeting composition described in any one of clauses 2-10, an isolated polynucleotide described in clause 11, a vector described in any one of clauses 12-20, a composition described in any one of clauses 23-34, or a modified adeno-associated viral vector described in clause 36.

[0205] Clause 38. The method of clause 37, wherein said correction of the mutant dystrophin gene comprises nuclease-mediated non-homologous end joining or homology-directed repair.

[0206] Clause 39. A method for genome editing a mutant dystrophin gene in a subject, the method comprising administering to the subject a genome editing composition comprising a polynucleotide sequence encoding the Cpf1 gRNA described in clause 1, a DNA targeting composition described in any one of clauses 2 to 10, an isolated polynucleotide described in clause 11, a vector described in any one of clauses 12 to 20, a composition described in any one of clauses 23 to 34, or a modified adeno-associated viral vector described in clause 36.

[0207] Clause 40. The method of clause 39, wherein the genome editing composition is administered to the subject intramuscularly, intravenously, or a combination thereof.

[0208] Clause 41. The method of clause 39 or clause 40, wherein the genome editing comprises nuclease-mediated non-homologous end joining or homology-directed repair.

[0209] Clause 42. A method of treating a subject in need thereof having a mutated dystrophin gene, the method comprising administering to the subject a polynucleotide sequence encoding the Cpf1 gRNA described in clause 1, a DNA targeting composition described in any one of clauses 2-10, an isolated polynucleotide described in clause 11, a vector described in any one of clauses 12-20, a composition described in any one of clauses 23-34, or a modified adeno-associated viral vector described in clause 36.

[0210] Clause 43. A method of correcting a mutant dystrophin gene in a cell, comprising administering to the cell (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 1. A method for correcting mutant dystrophin in a cell, wherein the gRNA comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the vector is configured to form first and second double-stranded breaks in first and second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene.

[0211] Clause 44. The method of clause 43, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0212] Clause 45. The method of clause 43 or clause 44, wherein the mutant dystrophin gene comprises a premature stop codon, a disrupted reading frame, an ectopic splice acceptor site, or an ectopic splice donor site.

[0213] Clause 46. The method of clause 45, wherein said mutant dystrophin gene contains a premature stop codon and a frameshift mutation resulting in a truncated gene product.

[0214] Clause 47. The method of clause 43 or clause 44, wherein the mutant dystrophin gene comprises a deletion of one or more exons that disrupts the reading frame.

[0215] Clause 48. The method of any one of clauses 43 to 47, wherein the correction of the mutant dystrophin gene comprises deleting a premature stop codon, correcting a disrupted reading frame, or modulating splicing by disrupting a splice acceptor site or disrupting a splice donor sequence.

[0216] 49. The method of claim 48, wherein the correction of the mutant dystrophin gene comprises a deletion of exon 51.

[0217] Clause 50. The method of any one of clauses 43 to 49, wherein the correction of the mutant dystrophin gene comprises nuclease-mediated non-homologous end joining or homology-directed repair.

[0218] Item 51. The method of any one of items 43 to 50, wherein the cells are myoblasts.

[0219] Item 52. The method of any one of items 43 to 51, wherein the cells are derived from a subject suffering from Duchenne muscular dystrophy.

[0220] Clause 53. A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject (a) a first vector comprising a polynucleotide sequence encoding a first Cpf1 guide RNA (gRNA) and a first Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), and (b) a second vector comprising a polynucleotide sequence encoding a second Cpf1 gRNA and a second Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123), wherein the first Cpf1 gRNA and the second Cpf1 A method of treating a subject, wherein the gRNA comprises a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 36-64, 71-119, or a complement thereof, and the first vector and the second vector are configured to form first and second double-stranded breaks in first and second introns, respectively, adjacent to exon 51 of the human dystrophin gene, thereby deleting a segment including exon 51 in the dystrophin gene.

[0221] Clause 54. The method of clause 53, wherein the first Cpf1 gRNA and the second Cpf1 gRNA are selected from the group consisting of: (i) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 54, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 62; (ii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 55, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 63; and (iii) a first Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 56, and a second Cpf1 gRNA comprising the polynucleotide sequence set forth in SEQ ID NO: 61.

[0222] Clause 55. The method of any one of clauses 53 or 54, wherein the subject is afflicted with Duchenne muscular dystrophy.

[0223] Clause 56. The method of any one of clauses 53 to 55, wherein the first vector and the second vector are administered to the subject intramuscularly, intravenously, or a combination thereof.

[0224] Item 57. A Cpf1 guide RNA (gRNA) targeting an enhancer of the B-cell lymphoma / leukemia 11A (BCL11a) gene, comprising a polynucleotide sequence corresponding to at least one of SEQ ID NOs: 65-70, or a complement thereof.

[0225] Clause 58. A method for disrupting an enhancer of B-cell lymphoma / leukemia 11A gene in a cell, said method comprising administering to said cell at least one Cpf1 gRNA and Cpf1 endonuclease described in clause 57.

Claims

1. A Cpf1 guide RNA (gRNA) that targets an enhancer of the B-cell lymphoma / leukemia 11A (BCL11a) gene and is encoded by a polynucleotide sequence comprising a sequence selected from SEQ ID NOs:64-70, or a complement thereof.

2. A DNA targeting composition comprising a Cpf1 endonuclease and at least one Cpf1 gRNA described in claim 1.

3. The DNA targeting composition of claim 2, wherein the Cpf1 endonuclease recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

4. The Cpf1 endonuclease is capable of inhibiting the activity of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albenzis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium MC2017 2, bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens eligens, Moraxella bovoculi 237, Leptospira inadais, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae.

5. 5. The DNA targeting composition of claim 2, wherein the Cpf1 endonuclease is derived from Lachnospiraceae bacterium ND2006 (LbCpf1) or Acidaminococcus (AsCpf1).

6. The DNA targeting composition of any one of claims 2 to 5, wherein the Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO:124 or SEQ ID NO:

125.

7. 10. An isolated polynucleotide comprising a polynucleotide sequence encoding the Cpf1 gRNA of claim 1 or the DNA targeting composition of any one of claims 2 to 6.

8. A vector comprising a polynucleotide sequence encoding the Cpf1 gRNA of claim 1, a DNA targeting composition of any one of claims 2 to 6, or an isolated polynucleotide of claim 7.

9. The vector of claim 8 , further comprising a polynucleotide sequence encoding a Cpf1 endonuclease.

10. The vector according to claim 8 or 9, wherein the vector is a viral vector.

11. The vector of claim 10 , wherein the vector is an adeno-associated virus (AAV) vector.

12. The vector of any one of claims 8 to 11, wherein the vector comprises a tissue-specific promoter operably linked to a polynucleotide sequence encoding a Cpf1 gRNA and / or the Cpf1 endonuclease.

13. The vector of claim 12 , wherein the tissue-specific promoter is a muscle-specific promoter.

14. A cell comprising the Cpf1 gRNA of claim 1, a polynucleotide sequence encoding the DNA targeting composition of any one of claims 2 to 6, an isolated polynucleotide of claim 7, or a vector of any one of claims 8 to 13.

15. A kit comprising the Cpf1 gRNA of claim 1, a polynucleotide sequence encoding the DNA targeting composition of any one of claims 2 to 6, an isolated polynucleotide of claim 7, a vector of any one of claims 8 to 13, or a cell of claim 14.

16. 1. A composition for disrupting an enhancer of the B-cell lymphoma / leukemia 11A gene in a cell, the composition comprising a vector comprising a polynucleotide sequence encoding a Cpf1 guide RNA (gRNA) and a polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123); wherein the Cpf1 gRNA is encoded by a polynucleotide sequence comprising at least one of SEQ ID NOs: 64-70, or a complement thereof.

17. 17. The composition of claim 16, wherein the Cpf1 endonuclease is a CPF1 endonuclease from Lachnospiraceae bacterium ND2006 (LbCpf1) or Acidaminococcus (AsCpf1).

18. 18. The composition of claim 16 or 17, wherein the Cpf1 endonuclease is encoded by a polynucleotide sequence comprising SEQ ID NO:124 or SEQ ID NO:

125.

19. The composition according to any one of claims 16 to 18, wherein the vector is a viral vector.

20. 20. The composition of claim 19, wherein the vector is an adeno-associated virus (AAV) vector.

21. The composition of claim 20, wherein the AAV vector is an AAV8 vector or an AAV9 vector.

22. The composition according to any one of claims 16 to 21, wherein the BCL11a gene is a human BCL11a gene.

23. A composition according to any one of claims 16 to 22 for use in medicine.

24. A composition according to any one of claims 16 to 22 for use in the treatment of sickle cell anemia.

25. A cell comprising the composition according to any one of claims 16 to 24.

26. A modified adeno-associated virus (AAV) vector for enhancer disruption of B-cell lymphoma / leukemia 11A gene, comprising a first polynucleotide sequence encoding a Cpf1 gRNA described in claim 1 and a second polynucleotide sequence encoding a Cpf1 endonuclease that recognizes a protospacer adjacent motif (PAM) of TTTA (SEQ ID NO: 120), TTTG (SEQ ID NO: 121), TTTC (SEQ ID NO: 122), or TTTT (SEQ ID NO: 123).

27. A composition for enhancer disruption of B-cell lymphoma / leukemia 11A gene comprising a polynucleotide sequence encoding the Cpf1 gRNA of claim 1, or a DNA targeting composition of any one of claims 2-6, or an isolated polynucleotide of claim 7, or a vector of any one of claims 8-13.

28. 28. The composition of claim 27, wherein the enhancer disruption of the B cell lymphoma / leukemia 11A gene comprises nuclease-mediated non-homologous end joining or homology-directed repair.

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