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CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors

2020; Cell Press; Volume: 181; Issue: 1 Linguagem: Inglês

10.1016/j.cell.2020.03.023

ISSN

1097-4172

Autores

Dan Wang, Feng Zhang, Guangping Gao,

Tópico(s)

Virus-based gene therapy research

Resumo

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments. The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments. Numerous human diseases arise from mutations that diminish or damage gene products. Gene therapy as a strategy to treat genetic diseases was formally proposed in 1972 (Friedmann and Roblin, 1972Friedmann T. Roblin R. Gene therapy for human genetic disease?.Science. 1972; 175: 949-955Crossref PubMed Google Scholar), introducing the concept that “genes can be medicine.” In the ensuing decades, implementation of this medical concept was met with initial excitement, serious setbacks, resurgence of interest, and more recently, clinical successes (Dunbar et al., 2018Dunbar C.E. High K.A. Joung J.K. Kohn D.B. Ozawa K. Sadelain M. Gene therapy comes of age.Science. 2018; 359: eaan4672Crossref PubMed Scopus (223) Google Scholar, High and Roncarolo, 2019High K.A. Roncarolo M.G. Gene Therapy.N. Engl. J. Med. 2019; 381: 455-464Crossref PubMed Scopus (28) Google Scholar). Despite these successes, however, delivering a functional gene copy to replace a mutated one is not a perfect solution for many diseases. For example, an exogenous gene copy lacks many regulatory elements that are important for endogenous gene expression and function. In addition, for gain-of-function pathogenic mutations, simply supplying a wild-type copy of the gene is ineffective. These and other limitations can be theoretically addressed by directly “editing” a mutated gene, thereby restoring gene function in its natural context. Indeed, many of our cells carry out something like this thousands of times a day, using a variety of DNA repair mechanisms that guard genome integrity in response to DNA damage. Similar to DNA damage, a targeted double-stranded break (DSB) can also trigger these cellular repair mechanisms, mainly, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), which can potentially introduce DNA sequence changes during repair of the DSB. HDR, which is a templated process, allows for the introduction of specific DNA changes, a phenomenon that has been leveraged to achieve insertion of new DNA sequences (Thomas and Capecchi, 1987Thomas K.R. Capecchi M.R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1670) Google Scholar). HDR-mediated insertion, however, requires the presence of a correct template (which is missing in most natural cases of genetic mutations) and is generally less efficient than NHEJ. More efficient methods for gene editing arose from the observation that a targeted DSB generated by an endonuclease can dramatically stimulate HDR in eukaryotic cells (Rouet et al., 1994Rouet P. Smih F. Jasin M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.Proc. Natl. Acad. Sci. USA. 1994; 91: 6064-6068Crossref PubMed Scopus (394) Google Scholar, Smih et al., 1995Smih F. Rouet P. Romanienko P.J. Jasin M. Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells.Nucleic Acids Res. 1995; 23: 5012-5019Crossref PubMed Google Scholar). This observation spurred a quest for programmable and efficient endonucleases (Urnov, 2018Urnov F.D. Genome Editing B.C. (Before CRISPR): Lasting Lessons from the “Old Testament”.CRISPR J. 2018; 1: 34-46Crossref PubMed Google Scholar), leading to the development of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short-palindromic repeat (CRISPR)-associated (Cas) proteins (Gaj et al., 2013Gaj T. Gersbach C.A. Barbas 3rd, C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (1791) Google Scholar, Hsu et al., 2014Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (2357) Google Scholar), the latter of which has been most widely adopted in research. Fundamental to the popularity of Cas nucleases is that they are guided by a short RNA sequence that recognizes the target DNA sequence through Watson-Crick base pairing (Brouns et al., 2008Brouns S.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J. Snijders A.P. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes.Science. 2008; 321: 960-964Crossref PubMed Scopus (1212) Google Scholar, Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (5388) Google Scholar, Garneau et al., 2010Garneau J.E. Dupuis M.E. Villion M. Romero D.A. Barrangou R. Boyaval P. Fremaux C. Horvath P. Magadan A.H. Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA..Nature. 2010; 468: 67-71Crossref PubMed Scopus (1023) Google Scholar, Gasiunas et al., 2012Gasiunas G. Barrangou R. Horvath P. Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proceedings of the National Academy of Sciences of the United States of America. 2012; 109: E2579-E2586Crossref PubMed Scopus (0) Google Scholar), whereas the other enzymes rely on protein-DNA interactions to achieve target specificity. Following the demonstration of CRISPR-Cas9-mediated genome editing (Cong et al., 2013Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (6618) Google Scholar, Mali et al., 2013Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (4595) Google Scholar), CRISPR-based technology has rapidly advanced, benefitting greatly from a collective endeavor to characterize, improve, expand, and share the CRISPR-based molecular toolbox (Doudna, 2020Doudna J.A. The promise and challenge of therapeutic genome editing.Nature. 2020; 578: 229-236Crossref PubMed Scopus (1) Google Scholar, Doudna and Charpentier, 2014Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (2080) Google Scholar, Zhang, 2019Zhang F. Development of CRISPR-Cas systems for genome editing and beyond.Q. Rev. Biophys. 2019; 51: 31Google Scholar). In addition to accelerating basic research, CRISPR-based technology also holds enormous potential as a therapeutic, offering an approach to permanently correct disease-causing mutations. Deploying CRISPR-based therapeutics directly into the human body holds great promise for treating numerous diseases. Although the CRISPR-based toolbox enables diverse operations ranging from DNA and RNA editing to gene expression modulation, delivery remains a bottleneck for therapy development. Currently, adeno-associated virus (AAV) vector is the leading platform for in vivo gene therapy delivery (Wang et al., 2019Wang D. Tai P.W.L. Gao G. Adeno-associated virus vector as a platform for gene therapy delivery.Nat. Rev. Drug Discov. 2019; 18: 358-378Crossref PubMed Scopus (65) Google Scholar). AAV is safe, capable of delivering its single-stranded DNA (ssDNA) vector genome to various tissues and cell types, and only mildly immunogenic within a wide range of dosing regimens. Although the vector genome largely remains episomal inside host cells, it is stabilized through concatemerization and circularization to mediate long-term transgene expression in post-mitotic cells, leading to durable therapeutic efficacy. The successes of AAV vectors in delivering gene therapies to disease animal models and patients propelled their adoption for in vivo delivery of CRISPR-based therapeutics. In this review, we focus on recent advances in CRISPR-based therapeutic strategies and in vivo delivery of CRISPR machinery using AAV vectors. We also discuss the limitations of using AAV vectors to deliver CRISPR-based therapeutics. Although the lessons learned from AAV gene therapy are generally applicable to delivering CRISPR-based tools, many challenges are unique to this new class of cargoes and call for distinct solutions from both the CRISPR and AAV fields to fully unleash the power of CRISPR-based therapeutic gene editing. Gene disruption by NHEJ. Typically, DSBs introduced with CRISPR are repaired via NHEJ, an efficient and prevalent DNA repair mechanism in human cells. NHEJ ligates two broken DNA ends together at the break site (Chapman et al., 2012Chapman J.R. Taylor M.R. Boulton S.J. Playing the end game: DNA double-strand break repair pathway choice.Mol. Cell. 2012; 47: 497-510Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). This process of targeted cleavage and repair can take place repeatedly until an insertion or deletion (indel) occurs that prevents further recognition of the target site by the nuclease. An indel mutation in a protein-coding gene can cause frameshifting or exon skipping, thereby disrupting gene function (Figure 1A). This seemingly disruptive gene editing approach has several therapeutic potentials. For example, the PCSK9 gene encodes an enzyme that binds to the cell surface receptor for low-density lipoprotein (LDLR) and triggers the lysosomal degradation of LDLR. When PCSK9 is diminished by gene editing, LDLR can return to the cell surface and continue to remove LDL, thereby lowering cholesterol levels (Ding et al., 2014Ding Q. Strong A. Patel K.M. Ng S.L. Gosis B.S. Regan S.N. Cowan C.A. Rader D.J. Musunuru K. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing.Circ. Res. 2014; 115: 488-492Crossref PubMed Scopus (233) Google Scholar, Ran et al., 2015Ran F.A. Cong L. Yan W.X. Scott D.A. Gootenberg J.S. Kriz A.J. Zetsche B. Shalem O. Wu X. Makarova K.S. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1088) Google Scholar). Similarly, loss of CCR5—a co-receptor exploited by HIV to infect T cells—confers HIV resistance on edited T cells (Xu et al., 2019bXu L. Wang J. Liu Y. Xie L. Su B. Mou D. Wang L. Liu T. Wang X. Zhang B. et al.CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia.The New England journal of medicine. 2019; 381: 1240-1247Crossref PubMed Scopus (0) Google Scholar, Xu et al., 2017Xu L. Yang H. Gao Y. Chen Z. Xie L. Liu Y. Liu Y. Wang X. Li H. Lai W. et al.CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo..Molecular therapy: the journal of the American Society of Gene Therapy. 2017; 25: 1782-1789Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Another application of gene disruption is silencing dominant negative mutations. Through careful design of the guide RNA, the mutant allele can be differentially disrupted while the normal one is preserved (Bakondi et al., 2016Bakondi B. Lv W. Lu B. Jones M.K. Tsai Y. Kim K.J. Levy R. Akhtar A.A. Breunig J.J. Svendsen C.N. et al.In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.Molecular therapy: the journal of the American Society of Gene Therapy. 2016; 24: 556-563Abstract Full Text Full Text PDF PubMed Google Scholar, György et al., 2019György B. Nist-Lund C. Pan B. Asai Y. Karavitaki K.D. Kleinstiver B.P. Garcia S.P. Zaborowski M.P. Solanes P. Spataro S. et al.Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.Nat. Med. 2019; 25: 1123-1130Crossref PubMed Scopus (6) Google Scholar, Rabai et al., 2019Rabai A. Reisser L. Reina-San-Martin B. Mamchaoui K. Cowling B.S. Nicot A.S. Laporte J. Allele-Specific CRISPR/Cas9 Correction of a Heterozygous DNM2 Mutation Rescues Centronuclear Myopathy Cell Phenotypes.Mol. Ther. Nucleic Acids. 2019; 16: 246-256Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) (Figure 1B). Predictable editing with a single cut. Although the indel mutations generated by Cas9-initiated NHEJ repair are heterogenous, the mutation spectrum is not random but reproducible and dependent on the target site and sequence context (van Overbeek et al., 2016van Overbeek M. Capurso D. Carter M.M. Thompson M.S. Frias E. Russ C. Reece-Hoyes J.S. Nye C. Gradia S. Vidal B. et al.DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.Mol. Cell. 2016; 63: 633-646Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). A data-trained machine-learning model was developed to predict the types and frequencies of Cas9-mediated small indels in human and mouse cells with high accuracy (Shen et al., 2018Shen M.W. Arbab M. Hsu J.Y. Worstell D. Culbertson S.J. Krabbe O. Cassa C.A. Liu D.R. Gifford D.K. Sherwood R.I. Predictable and precise template-free CRISPR editing of pathogenic variants.Nature. 2018; 563: 646-651Crossref PubMed Scopus (69) Google Scholar). This model identifies human pathogenic variants that, following Cas9 cleavage, can be corrected by the predicted predominant indels. This template-free editing method was used to correct frameshift mutations and microduplication mutations in human cells (Iyer et al., 2019Iyer S. Suresh S. Guo D. Daman K. Chen J.C.J. Liu P. Zieger M. Luk K. Roscoe B.P. Mueller C. et al.Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.Nature. 2019; 568: 561-565Crossref PubMed Scopus (12) Google Scholar, Shen et al., 2018Shen M.W. Arbab M. Hsu J.Y. Worstell D. Culbertson S.J. Krabbe O. Cassa C.A. Liu D.R. Gifford D.K. Sherwood R.I. Predictable and precise template-free CRISPR editing of pathogenic variants.Nature. 2018; 563: 646-651Crossref PubMed Scopus (69) Google Scholar). Another class of predictable editing is targeting a splicing signal to induce exon skipping (Amoasii et al., 2017Amoasii L. Long C. Li H. Mireault A.A. Shelton J.M. Sanchez-Ortiz E. McAnally J.R. Bhattacharyya S. Schmidt F. Grimm D. et al.Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy.Sci. Transl. Med. 2017; 9: eaan8081Crossref PubMed Scopus (53) Google Scholar, Long et al., 2018Long C. Li H. Tiburcy M. Rodriguez-Caycedo C. Kyrychenko V. Zhou H. Zhang Y. Min Y.L. Shelton J.M. Mammen P.P.A. et al.Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing.Sci Adv. 2018; 4: eaap9004Crossref PubMed Scopus (50) Google Scholar). Compared with a 2-cut approach (see below), using one single-guide RNA (sgRNA) to destroy an exonic splicing enhancer, a splicing acceptor site, or a splicing donor site offers a simplified therapeutic design. Precise mutation repair by HDR. Although precise correction of DNA mutations by HDR is an intuitive therapeutic approach (Figure 1C), this DNA repair mechanism is inefficient in human cells, especially in post-mitotic cells like myofibers and neurons (Chapman et al., 2012Chapman J.R. Taylor M.R. Boulton S.J. Playing the end game: DNA double-strand break repair pathway choice.Mol. Cell. 2012; 47: 497-510Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). Furthermore, it requires the co-delivery of a repair donor template that carries homology arms matching the targeted locus, which complicates the therapy. Nevertheless, this is a powerful therapeutic strategy, especially in cases where a small number of corrected cells can improve symptoms or in cases where regenerative tissues are targeted. For example, hereditary tyrosinemia type I (HT-1) is a metabolic disease caused by mutations in the FAH gene, which is expressed in actively dividing hepatocytes. In a mouse model of HT-1, delivery of Cas9, sgRNA, and a donor template to the liver corrected a disease-causing Fah mutation in some hepatocytes, which then had a growth advantage compared to unedited cells, ultimately driving rescue of the disease phenotype (Yin et al., 2014Yin H. Xue W. Chen S. Bogorad R.L. Benedetti E. Grompe M. Koteliansky V. Sharp P.A. Jacks T. Anderson D.G. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.Nat. Biotechnol. 2014; 32: 551-553Crossref PubMed Scopus (552) Google Scholar, Yin et al., 2016Yin H. Song C.Q. Dorkin J.R. Zhu L.J. Li Y. Wu Q. Park A. Yang J. Suresh S. Bizhanova A. et al.Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.Nat. Biotechnol. 2016; 34: 328-333Crossref PubMed Scopus (379) Google Scholar). Targeted gene insertion. Another application of HDR-based gene editing is targeted insertion of exogenous DNA sequence into the genome (Figure 1D). This is best exemplified by integrating a chimeric antigen receptor (CAR) gene into isolated T cells; after being infused back into the patient, these armed T cells are capable of recognizing and killing tumor cells specified by the CAR (Bailey and Maus, 2019Bailey S.R. Maus M.V. Gene editing for immune cell therapies.Nat. Biotechnol. 2019; 37: 1425-1434Crossref PubMed Scopus (15) Google Scholar). Using CRISPR to achieve targeted integration of the CAR by HDR can achieve concomitant disruption of the T-cell receptor α constant (TRAC) locus, resulting in engineered CAR-T cells that may be more amenable to allogeneic infusion (Eyquem et al., 2017Eyquem J. Mansilla-Soto J. Giavridis T. van der Stegen S.J. Hamieh M. Cunanan K.M. Odak A. Gönen M. Sadelain M. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.Nature. 2017; 543: 113-117Crossref PubMed Scopus (383) Google Scholar). In addition to HDR-based approaches, homology-independent targeted integration (HITI) was developed to utilize the more robust NHEJ repair pathway for gene integration, which the authors reported achieved efficient rescue of a disease phenotype in mice (Suzuki et al., 2016Suzuki K. Tsunekawa Y. Hernandez-Benitez R. Wu J. Zhu J. Kim E.J. Hatanaka F. Yamamoto M. Araoka T. Li Z. et al.In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.Nature. 2016; 540: 144-149Crossref PubMed Scopus (353) Google Scholar). Notably, a single AAV vector design can incorporate elements allowing for both HDR-mediated gene insertion and HITI to achieve efficient genetic correction (Ohmori et al., 2017Ohmori T. Nagao Y. Mizukami H. Sakata A. Muramatsu S.I. Ozawa K. Tominaga S.I. Hanazono Y. Nishimura S. Nureki O. Sakata Y. CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures haemophilia B mice.Sci. Rep. 2017; 7: 4159Crossref PubMed Scopus (43) Google Scholar). Large-scale DNA editing. CRISPR is inherently capable of multiplex editing, and this natural feature has been leveraged to achieve deletion of large sections of DNA. By introducing two guide RNAs targeting separate sites, fragments as large as several megabase pairs (Mbp) can be excised from the genome (Figure 1E). This approach can remove deleterious mutations while maintaining the open reading frame by deleting one or more exons. This strategy is particularly well suited for Duchene muscular dystrophy (DMD), which is caused by several different mutations, all of which can be treated with this strategy because truncated dystrophin protein with internal deletions is partially functional (England et al., 1990England S.B. Nicholson L.V. Johnson M.A. Forrest S.M. Love D.R. Zubrzycka-Gaarn E.E. Bulman D.E. Harris J.B. Davies K.E. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin.Nature. 1990; 343: 180-182Crossref PubMed Scopus (432) Google Scholar). In a DMD mouse model carrying a nonsense mutation in the dystrophin gene in exon 23, AAV delivery to the muscle of Cas9 and a pair of sgRNAs flanking exon 23 mediated deletion of this exon. Splicing between exon 22 and exon 24 preserved the normal reading frame, generating truncated but functional dystrophin that rescued the disease phenotype (Long et al., 2016Long C. Amoasii L. Mireault A.A. McAnally J.R. Li H. Sanchez-Ortiz E. Bhattacharyya S. Shelton J.M. Bassel-Duby R. Olson E.N. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.Science. 2016; 351: 400-403Crossref PubMed Scopus (463) Google Scholar, Nelson et al., 2016Nelson C.E. Hakim C.H. Ousterout D.G. Thakore P.I. Moreb E.A. Castellanos Rivera R.M. Madhavan S. Pan X. Ran F.A. Yan W.X. et al.In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.Science. 2016; 351: 403-407Crossref PubMed Google Scholar, Tabebordbar et al., 2016Tabebordbar M. Zhu K. Cheng J.K.W. Chew W.L. Widrick J.J. Yan W.X. Maesner C. Wu E.Y. Xiao R. Ran F.A. et al.In vivo gene editing in dystrophic mouse muscle and muscle stem cells.Science. 2016; 351: 407-411Crossref PubMed Scopus (517) Google Scholar). Another example of this therapeutic strategy is removal of the IVS26 mutation in the CEP290 gene, which causes Leber congenital amaurosis type 10 (LCA10) (Maeder et al., 2019Maeder M.L. Stefanidakis M. Wilson C.J. Baral R. Barrera L.A. Bounoutas G.S. Bumcrot D. Chao H. Ciulla D.M. DaSilva J.A. et al.Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.Nat. Med. 2019; 25: 229-233Crossref PubMed Scopus (61) Google Scholar). This point mutation is in intron 26 and causes aberrant splicing. Using a similar AAV-based approach for eye delivery in a humanized mouse model carrying the IVS26 mutation, a pair of sgRNAs was used to guide Cas9 to excise a segment of intron 26 that contains the mutation, restoring normal splicing between exon 26 and exon 27. The editing efficiency met the targeted therapeutic threshold and was translatable to nonhuman primates (Maeder et al., 2019Maeder M.L. Stefanidakis M. Wilson C.J. Baral R. Barrera L.A. Bounoutas G.S. Bumcrot D. Chao H. Ciulla D.M. DaSilva J.A. et al.Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.Nat. Med. 2019; 25: 229-233Crossref PubMed Scopus (61) Google Scholar). Based on these preclinical results, a phase 1/2 clinical trial for the treatment of LCA10 is currently open (ClinicalTrials.gov; identifier: NCT03872479), and it is expected to be the first in vivo clinical application of CRISPR-based gene editing. At an even larger scale, recessive compound heterozygous mutations—different mutations damaging the same gene—can be corrected by allelic exchange mediated by DNA cuts at two homologous chromosomes and inter-homologue translocation (Wang et al., 2018Wang D. Li J. Song C.Q. Tran K. Mou H. Wu P.H. Tai P.W.L. Mendonca C.A. Ren L. Wang B.Y. et al.Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice.Nat. Biotechnol. 2018; 36: 839-842Crossref PubMed Scopus (7) Google Scholar) (Figure 1F). Although compound heterozygous mutations are prevalent in patients, application of allelic exchange in a broad range of diseases will require enhancing the efficiency of allelic exchange. RNA targeting. Following the discovery and development of RNA-targeting Cas enzymes for use in human cells (Abudayyeh et al., 2016Abudayyeh O.O. Gooteberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B.T. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector..Science. 2016; 353: aaf5573Crossref PubMed Scopus (0) Google Scholar, Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (311) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.Cell. 2018; 173: 665-676.e614Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.Mol. 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Compared with editing at the level of the genome, gene knockdown at the RNA level is a potentially reversible therapeutic approach. In addition, RNA targeting enzymes, like Cas13, offer more flexible target selection than Cas9 and more specific target degradation than RNA interference (RNAi) (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (311) Google Scholar). Repeated intratumoral delivery of Cas13a and guide RNA targeting a mutant KRAS transcript in the form of ribonucleoprotein (RNP) was shown to slow tumor growth in a xenograft mouse model (Zhao et al., 2018Zhao X. Liu L. Lang J. Cheng K. Wang Y. Li X. Shi J. Wang Y. Nie G. A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment.Cancer Lett. 2018; 431: 171-181Crossref PubMed Scopus (12) Google Scholar). To achieve long-lasting in vivo therapeutic effects by targeting RNA, AAV vectors are an advantageous delivery platform for Cas13-based tools because they allow for durable expression of the editing system. CRISPR interference and activation (CRISPRi/a). In addition to its use for gene editing, Cas9 has been repurposed as an RNA-guided DNA binding domain by introducing mutations in the RuvC and HNH nuclease domains (Bikard et al., 2013Bikard D. Jiang W. Samai P. Hochschild A. Zhang F. Marraffini L.A. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Nucleic Acids Res. 2013; 41: 7429-7437Crossref PubMed Scopus (453) Google Scholar, Qi et al., 2013Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013; 152: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (1848) Google Scholar). In mammalian cells, robust gene repression was achieved by fusing a nuclease-deactivated Cas9 (dCas9) to a repressor domain like the Krüppel-associated box (KRAB) domain and targeting it to the transcription start site (Gilbert et al., 2014Gilbert L.A. Horlbeck M.A. Adamson B. Villalta J.E. Chen Y. Whitehead E.H. Guimaraes C. Panning B. Ploegh H.L. Bassik M.C. et al.Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.Cell. 2014; 159: 647-661Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, Gilbert et al., 2013Gilbert L.A. Larson M.H. Morsut L. Liu Z. Brar G.A. Torres S.E. Stern-Ginossar N. Brandman O. Whitehead E.H. Doudna J.A. et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell. 2013; 154: 442-451Abstract Full Text Full Text PDF PubMed Scopus (1413) Google Scholar) (Figure 2A). AAV delivery of CRISPRi targeting Pcsk9 in mouse liver effectively silenced transcription and lowered serum PCSK9 and cholesterol levels (Thakore et al., 2018Thakore P.I. Kwon J.B. Nelson C.E. Rouse D.C. Gemberling M.P. Oliver M.L. Gersbach C.A. RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.Nat. Commun. 2018; 9: 1674Crossref PubMed Scopus (35) Google Scholar). Replacing the KRAB domain with a gene activating domain, like VP64, converts a gene repressor to a gene activator (Cheng et al., 2013Cheng A.W. Wang H. Yang H. Shi

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