- Email: [email protected]
CRISPR–Cas Gene Editing for Neurological Disease
C H A P T E R
18 CRISPR–Cas Gene Editing for Neurological Disease Bryan P. Simpson*,†, Beverly L. Davidson*,† *
The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States †The Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, United States
INTRODUCTION Many neurological diseases are caused by genetic mutations, which means they are amenable to various gene therapy approaches. Such approaches include gene augmentation to replace the mutant gene, or gene knockdown to inhibit expression of a dominantly acting disease gene. Therapeutic modalities also include going after the mutant protein, either by replacing it (e.g., enzyme replacement therapy) or by inhibiting it (e.g., introducing antibodies to remove toxic gene products). Recently, gene editing has been added to the toolbox of methodologies to reduce the disease gene burden. Among gene editing techniques is the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated nuclease (Cas) gene editing technology, referred to as CRISPR–Cas. This modality offers the flexibility to edit, replace, and modify specific loci in the genome with simplicity and ease in the laboratory, which makes it a useful tool to knockout and knock-in genes, to activate or silence gene expression, and to create model systems.1 The CRISPR–Cas system has been well characterized since its rise to fame and is now moving toward therapeutic development, having shown promise in disease models.2, 3 This chapter highlights the studies and findings supporting the feasibility and utility of therapeutic CRISPR–Cas gene editing as it applies to neurological disease.
CRISPR–Cas GENE EDITING IN MAMMALIAN SYSTEMS CRISPR is a bacterial adaptive immune system that targets foreign nucleic acid sequences (Fig. 1).4, 5 Bacteria use this system to defend against infections by incorporating short
Nervous System Drug Delivery https://doi.org/10.1016/B978-0-12-813997-4.00018-9
365
# 2019 Elsevier Inc. All rights reserved.
366
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
FIG. 1 The CRISPR–Cas adaptive immune system in prokaryotes. The clustered regularly interspaced short palindromic repeat (CRISPR) locus is a genomic region that provides adaptive immunity against viral infections in prokaryotes. When foreign nucleic acid is introduced into the host cell, the Cas operon expresses CRISPR-associated proteins (i.e., Cas) to select and integrate a protospacer sequence into the CRISPR array. The CRISPR array expresses pre-CRISPR RNA (crRNA) that is further processed into mature crRNA. The Cas nuclease protein complexes with the crRNA, which guides it to the sequence-specific target for cleavage.
fragments of the foreign DNA within the CRISPR region of its genome. The CRISPR region consists of short, repetitive, palindromic spacer sequences, and sequences encoding Cas proteins.6 When the foreign DNA fragments are expressed, they behave as guide RNAs (gRNAs) to direct the Cas nuclease to the invading target, which results in cleavage of the foreign agent’s DNA.1 The fusion of CRISPR RNA (crRNA) and the trans-activating CRISPR RNA (tracrRNA) into a chimeric single-guide RNA (sgRNA) allows for site-specific gene editing in eukaryote genomes (Fig. 2).7, 8 The CRISPR type II system with the Streptococcus pyogenes Cas9 (SpCas9) nuclease is the most commonly used system, and scientists take advantage of it by designing an sgRNA that targets a specific locus, and expressing the sgRNA along with Cas9 in vitro or in vivo.9 Cas9 associates with the sgRNA, and the complex is targeted to the desired DNA sequence.2, 10, 11 The Cas9-sgRNA complex binds to the protospacer adjacent motif (PAM) sequence located next to the sgRNA target sequence, and the two enzymatic endonuclease domains of Cas9 (i.e., RuvC and HNH) cleave both strands of DNA to generate a doublestrand break (DSB).7, 12, 13 The DSB can be repaired by one of the two DNA repair mechanisms: nonhomologous end joining (NHEJ) or homology-directed repair (HDR).14 NHEJ, the predominant repair pathway in mammalian cells, is prone to error and can result in insertion or deletion (i.e., indel) of nucleotides. Indels can create frameshift mutations and downstream premature stop codons, leading to inactivation of the target gene.2, 11
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
CRISPR–Cas GENE EDITING IN MAMMALIAN SYSTEMS
367
FIG. 2 CRISPR–Cas9 gene editing in eukaryotes. The sequence-specific Cas9-sgRNA complex binds the protospacer adjacent motif (PAM), the protospacer sequence of the sgRNA anneals; meanwhile, the catalytic nuclease domains (i.e., RuvC and HNH) cleave the target DNA sequence to create a double-strand break (DSB). The errorprone, nonhomologous end-joining (NHEJ) repair pathway can create insertion and deletion (i.e., indel) mutations that can result in a frameshift, premature stop codon, and, ultimately, gene knockout. Alternatively, the homologydirected repair pathway can modify or correct a gene by knock-in when a donor DNA template is provided.
In addition to gene knockout and inactivation, targeted gene insertion is possible by CRISPR–Cas9 HDR-mediated knock-in of donor DNA sequence templates,15-17 which can be harnessed to correct loss-of-functions mutations.18 HDR may be less efficient and somewhat limited in terminally differentiated postmitotic cells such as neurons.16 However, NHEJ in neurons can occur, allowing for knock-in editing strategies. The CRISPR-Cpf1 system with RNA-guided Cpf1, a nuclease like Cas9, generates DSBs with overhangs; this in turn promotes NHEJ in neurons.19-21 Finally, CRISPR–Cas9 multiplexing is possible when multiple sgRNAs are used to target different genes and pathways simultaneously, or when more than one guide is required to modify a gene.15, 22-24 The CRISPR–Cas9 system also allows for transcriptional regulation to modulate gene expression without permanently editing the genome (Fig. 3). For this, a mutated, dead Cas9 (dCas9) that lacks enzymatic endonuclease activity is used.25 The dCas9-sgRNA complex binds the targeted DNA sequence without creating a DSB, and is used to silence or activate gene expression (processes known as CRISPR interference [CRISPRi] or CRISPR activation
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
368
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
FIG. 3
dCas9-mediated gene manipulation. Catalytically inactive dead Cas9 (dCas9) can functionally bind target DNA to impart transcriptional downregulation. Additionally, when dCas9 is fused with different effector domains, it is possible to enhance gene repression, activate gene expression, target epigenetic modifications, or edit DNA bases.
[CRISPRa], respectively). This enhanced gene regulation is achieved with effector protein domains fused to dCas9 to recruit epigenetic modifying factors.25-29 For CRISPRi, the transcriptional repressor domain Kr€ uppel associated box (KRAB) is fused to dCas9; for CRISPRa, the transcriptional activator domain VP64 is fused to dCas9.25, 30 Alternatively, sgRNA can be modified with an MS2 coat protein fused to an effector domain for robust transcriptional modulation.31, 32 CRISPR–Cas9 is delivered to cells using standard viral and nonviral methods. Currently, adeno-associated viral (AAV) vectors are the most widely used delivery method for in vivo delivery of CRISPR–Cas9; however, AAV packaging size is restricted to 4.7 kb, so a dualAAV vector system is required. One vector delivers the SpCas9, and the second vector delivers the sgRNA(s). Alternatively, Staphylococcus aureus Cas9 (SaCas9), a smaller Cas9 orthologue, can fit into a single AAV vector along with the sgRNAs.33 Because viral delivery and constitutive expression of CRISPR–Cas9 may increase off-target effects and the risk of an immune response to the foreign bacterial protein, short-lived ribonucleoprotein (RNP) complexes of sgRNA-Cas9 are attractive alternatives for gene editing. RNP delivery has been shown to be effective after direct injections into mouse cortex, striatum, and hippocampus.34 Additionally, in vivo RNP delivery of an allele-specific sgRNA-Cas9 complex resulted in correction of hearing loss in a dominant deafness-associated allele in the Tmc1 Beethoven mouse model.35 These studies support the potential for RNP delivery in neurological diseases to overcome the safety concern of sustained expression from viral delivery systems, or the need for more than one vector. The CRISPR–Cas9 system provides advantages over alternative genome editing tools, such as zinc-finger nucleases and transcription activator-like effector nucleases.36 Most notably, CRISPR–Cas9 is easily adaptable to any gene and does not require extensive optimization, although editing frequency can vary depending on the target sequence and the sgRNAs.37 In addition, CRISPR–Cas9 overcomes the limitations of RNA-targeting
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
MOVING CRISPR–Cas GENE EDITING TOWARD THE CLINIC
369
strategies for gain-of-function mutations, such as RNA interference and antisense oligonucleotides. These latter approaches, if delivered via nonviral means, require repeated treatments, and both can be prone to high off-target effects and cytotoxicity.38, 39 One major drawback of the CRISPR–Cas9 system is off-target effects due to sgRNA sequence homology to other genomic sites.40, 41 Cas9 can tolerate three to five mismatches, depending on the distribution and number of mismatched nucleotides.2, 13, 30, 42 Designing sgRNAs with low GC content and minimal homology to off-target sites, reducing the size of the sgRNA, and limiting the amount of Cas9 protein are all ways to minimize off-targets.12, 42, 43 The nickase Cas9, a mutated Cas9 with only one functionally enzymatic endonuclease domain, also reduces offtargets because it cuts only one strand of the DNA. However, the nickase Cas9 does require two sgRNAs targeting opposite DNA strands to create a DSB for generation of the desired indel.44, 45 Engineering advances in the CRISPR–Cas9 system have focused on optimizing editing efficiency, specificity, and Cas9 regulation. On-target specificity was improved by engineering an enhanced Cas9 and a high-fidelity Cas9; both were associated with reduced offtargeting.46, 47 An evolved SpCas9 variant, xCas9, is associated with improved specificity, low off-target activity, and broader PAM compatibility, all of which facilitate broader applicability.48 Another approach involves a synthetic crRNA to replace the wild-type crRNA, which reduces off-targets and enhances cleavage activity.49 Additionally, split-Cas9 systems have been developed to control and regulate Cas9 expression. In this scenario, Cas9 is expressed by two expression systems that can be induced to dimerize, and has editing activity similar to that of wild-type SpCas9.50-52 Finally, expression regulated by small molecules, light, or temperature have also been designed to control Cas9 activity.53
MOVING CRISPR–Cas GENE EDITING TOWARD THE CLINIC Disease-modifying treatments for neurological diseases are currently lacking, with most drugs targeting symptom management. Early CRISPR–Cas9 genome editing studies were used to create model systems, but advances in gene delivery of CRISPR–Cas9 technology now support the clinical utility of this modality for genome editing.54 For dominantly inherited neurological diseases, CRISPR–Cas9 gene editing could inactivate or transcriptionally downregulate disease-causing gain-of-function mutations. Additionally, CRISPR–Cas9 repair of mutant genes could correct autosomal recessive loss-of-function mutations.55, 56 Even more exciting is the recent development of CRISPR–Cas9 single-base editing strategies for correcting pathogenic point mutations (Fig. 3).57 Not long after CRISPR–Cas9 gene editing was performed in eukaryotic cells, in vivo editing of genes was investigated in the mammalian brain. The initial CRISPR–Cas9 gene editing studies in postmitotic neurons and the mammalian brain support the technology as a viable therapeutic option.58, 59 The first proof-of-principle study of CRISPR–Cas9 gene knockdown in neurons was done in embryonic mice,58 and CRISPR–Cas9 knockout of the N-methyl-D-aspartate receptor (NMDAR) subunit protein GluN1 was done in mouse hippocampal pyramidal neurons after in utero electroporation delivery. NMDAR currents were abolished, demonstrating functional in vivo knockdown of Grin1. Additionally, the authors transfected the Cas9/gRNA complex into postmitotic neurons in hippocampal slice cultures
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
370
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
from P6-P8 rats and showed similar reductions in NMDAR-dependent currents. Although the study did not demonstrate in vivo editing of neurons after CRISPR–Cas9 delivery into the brain, it was nonetheless a tremendous step forward. In other work, Mecp2 promoter-driven Cas9 and an sgRNA targeting the Mecp2 gene demonstrated high editing efficiency with concomitant changes in learning and behavior.59 The CRISPR–Cas9 modifications were stable after AAV delivery to adult mice hippocampal neurons. CRISPR–Cas9 multiplexing can also target multiple genes, suggesting the potential for treating polygenic disease such as psychiatric disorders;60 in fact, Cas9 coupled with three sgRNAs targeting Dnmt3a, Dnmt1, and Dnmt3b in the hippocampus induced behavioral alterations, setting the stage for in vivo CRISPR–Cas9 interrogation of pathways to address the roles of pathways in complex behavior, with possible adaptation to clinical problems. CRISPR–Cas9 application to correct a monogenic disease was first presented by three groups studying Duchenne muscular dystrophy in mice models.61-63 They showed editing and improvement in phenotypes after delivery of the editing machinery. CRISPR–Cas9 gene therapy was also studied in the setting of hereditary liver disease.64, 65 These pioneering researchers opened the door for ongoing in vivo CRISPR–Cas9 gene editing research and the application of this research toward treating neurological diseases. Currently, CRISPR–Cas9 technology is geared toward correcting monogenic diseases in patient cell lines and animal models of Alzheimer disease (AD), Parkinson disease, amyotrophic lateral sclerosis (ALS), and the repeat expansion disorders, such as fragile X syndrome (FXS), Huntington disease (HD), the autosomal dominant spinocerebellar ataxias (SCAs), and spinal and bulbar muscular atrophy (SBMA). Alzheimer disease is a major cause of morbidity and mortality in elderly individuals. Mutations in the presenilin genes PSEN1 and PSEN2 and in the amyloid precursor protein (APP) gene result in amyloid-associated pathology that leads to early-onset familial AD, and variants of APOE are risk factors for late-onset AD.43 Gyorgy and colleagues disrupted a mutant variant of APP in human fibroblasts, providing the first experimental evidence of CRISPR– Cas9 gene editing for treatment of familial forms of dominant AD.66 PSEN1 mutations were corrected in AD-patient-induced pluripotent stem cells (iPSCs) by HDR-mediated CRISPR– Cas9 when combined with homologous repair templates.67 Mutations in LRRK2 and SNCA are causes of autosomal dominant Parkinson disease.68 CRISPRi targeting the transcription start site of SNCA significantly reduced expression levels in vitro.69 Additionally, studies using dCas9-KRAB repression and dCas9-VPR activation showed that SNCA expression could be modulated in human iPSC-derived neurons. Polyglutamine (polyQ) repeat expansion disorders such as HD, Kennedy disease, and SCAs are caused by toxic gain-of-function mutations. HD is a neurodegenerative disease caused by an expanded CAG trinucleotide repeat in the huntingtin gene (i.e., HTT), which results in a mutant HTT protein with an expanded polyQ repeat. Several CRISPR–Cas9 approaches have been investigated to reduce expression of mutant HTT. Similar to findings in mouse models of muscular dystrophy, CRISPR-mediated HDR has been suggested as an exon-skipping strategy to target polyQ repeats.70, 71 Another approach is to reduce the size of the expanded CAG repeat. Using the CRISPR–Cas9 D10A nickase resulted in CAG/ CTG repeat contraction in a reporter vector.72 The problem with this approach is that other repeat-containing genes in the genome may be targeted. Replacement of the expanded polyQ repeat with a normal polyQ repeat by homologous recombination in iPSCs from HD patients
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
MOVING CRISPR–Cas GENE EDITING TOWARD THE CLINIC
371
improved disease phenotypes, suggesting the potential for a similar HDR-mediated approach using CRISPR–Cas9.73 However, the efficiency of an HDR-mediated approach may not be sufficient to correct disease in humans. Alternatively, CRISPRi targeting the transcription start site of HTT resulted in reduction of HTT mRNA and protein expression levels.69 Meanwhile, CRISPR-NHEJ-mediated deletion reduced mutant HTT expression in mesenchymal stem cells from the YAC128 mouse model of HD.74 CRISPR–Cas9 targeted deletion or contraction of the HTT polyQ repeat might not restore normal protein function, and whether complete knockout of both the mutant and wild-type HTT alleles will be tolerated remains unknown. To overcome these problems, allele-specific editing of the mutant allele was demonstrated with CRISPR–Cas9 by using single-nucleotide polymorphisms (SNPs) found in PAMs of the mutant allele. In one approach, two allelespecific sgRNAs targeting SNPs within PAMs on the mutant allele created a large deletion including the promoter (i.e., TSS), and expanded CAG from the mutant allele in patient fibroblasts, iPSCs, and neural progenitor cells.75 Ablation of mutant HTT mRNA and protein demonstrated the efficacy of allele-specific CRISPR–Cas9 without targeting the normal allele. A similar approach identified SNPs within PAMs flanking exon-1 of HTT in heterozygosity between the normal and mutant alleles.76 Allele-specific deletion of HTT exon-1 resulted in targeted mutant HTT mRNA and protein knockdown in human HD fibroblasts. In vivo allelespecific editing in the BACHD transgenic mouse model reduced human mutant HTT expression to 40%, a level known to provide therapeutic benefit by RNA interference or antisense oligonucleotides, demonstrating CRISPR–Cas9 in vivo efficacy in an HD mouse model for the first time. A nonallele-specific approach with sgRNAs flanking the CAG repeat reduced human mutant HTT in striatal neurons and improved motor deficits in the HD140Q-knock-in mouse model of HD, which shows the behavioral effects of mutant HTT reduction by in vivo CRISPR–Cas9.77 To address constitutive Cas9 expression, an off-switch was engineered and tested in HD patient-derived neurons and in a mouse brain.78 The “KamiCas9” contains an sgRNA that can reduce not only the target, but also the nuclease. Although this may reduce immune responses to the bacterial nuclease, further testing is required to determine the extent to which this also may alleviate unintended editing. The expansion of GGGGCC hexanucleotide (G4C2) repeats in the C9orf72 gene cause familial ALS and frontotemporal dementia.79, 80 Mutihac and colleagues (cited in Ref 18) corrected an expanded G4C2 repeat with HDR-mediated CRISPR–Cas9 in iPSCs from an individual with a C9orf72 mutation, and ameliorated disease-associated phenotypes such as protein aggregation and stress granule formation in differentiated motor neurons.18 In other work, mutations in microtubule-associated protein tau were corrected in iPSCs derived from individuals with frontotemporal dementia.81 Autosomal dominant mutations in SOD1 and FUS cause familial ALS. Single-nucleotide mutations in SOD1 and FUS were corrected by HDR-mediated CRISPR–Cas9 in iPSCs from individuals with familial ALS.82 Systemic AAV9 delivery of SaCas9 and an sgRNA targeting exon 2 of human SOD1 reduced mutant SOD1 expression by more than 2.5-fold in the spinal cord of neonatal G93A-SOD1 model mice of ALS,83 improving motor function and slowing muscle atrophy. Furthermore, the mice showed a delay in disease onset, more surviving neurons at end stage, and increased survival.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
372
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
Fragile X syndrome is a neurodevelopmental disorder that causes learning disabilities and cognitive impairment. FXS is a trinucleotide repeat disorder caused by CGG repeat expansion in the 50 UTR of the fragile X mental retardation 1 (FMR1) gene. This causes hypermethylation of FMR1 with transcriptional silencing and reduced fragile X mental retardation protein (FMRP) expression. CRISPR–Cas9 and an sgRNA targeting upstream of the FMR1 CGG repeat in patient FXS iPSCs resulted in deletion of CGG repeats and reactivation of FMR1 expression.84 In another study in FXS iPSCs, Cas9 was expressed along with two sgRNAs flanking the CGG repeat, resulting in precise, efficient removal of the repeat and increased FMR1 expression.85 An alternative approach targeted the hypermethylation of FMR1 with dCas9-Tet for demethylation of the CGG expansion at the FMR1 promoter to reactivate expression of FMR1 in FXS iPSCs.86 The edited iPSCs that were subsequently differentiated into neurons had wild-type phenotypes, and maintained FMR1 expression after engraftment into the mouse brain. Notably, in vivo CRISPR–Cas9 editing and reactivation of FMR1 has not yet been shown in vivo. A related disorder, fragile X-associated tremor/ataxia (FXTAS), is caused by smaller repeat expansions in this locus, referred to as premutation of FMR1, and is a lateonset neurodegenerative disease caused by increased FMR1 mRNA toxicity and decreased FMRP expression. Based on the CRISPR–Cas9 studies in FXS iPSCs, similar strategies to edit and correct FMR1 in FXTAS are worth pursuing.
CRISPR–Cas RNA TARGETING A new generation of CRISPR–Cas proteins is making it possible to study RNA biology and to develop RNA-targeting therapeutics. Programmable RNA-guided Cas13, an RNase protein, efficiently reduces target mRNAs, and a catalytically inactive dCas13 edited single-bases in mammalian cells.87, 88 RNA-targeting CRISPR–Cas9 methods can reduce mRNAs associated with HD, C9orf72-linked ALS, FTXTAS, SBMA, the SCAs, and myotonic dystrophy types 1 and 2 (DM1 and DM2). An engineered RNA-targeting SpCas9 (RCas9), which can bind RNA, eliminated exogenous expression of CUG, CCUG, CAG, and GGGGCC repeat expansion RNAs in cells, and endogenous CUG and CCUG expansions in DM1 and DM2 patient cells.89, 90 Targeting expanded CAG RNAs reduced disease-associated polyQ protein, supporting the approach in HD, SBMA, and the SCAs. The advantage of RNA targeting is that no permanent off-target effects at the DNA level have been observed, which makes it a potentially safer strategy. However, repeated dosing or sustained expression from vector systems will be required, raising the specter of off-targeting or immune responses to the foreign Cas proteins. Studying RNA-targeting CRISPR systems in vivo will be the next step toward an RNA-directed therapy for neurological diseases.
CRISPR TRANSCRIPTIONAL REGULATION IN THE MAMMALIAN BRAIN Transcriptional regulation with dCas9 has been widely used in vitro to modulate gene expression. The first proof-of-principle use of in vivo CRISPRi in the mammalian brain was done with a lentivirus expressing dCas9-KRAB to knockdown Syt1 expression in the dentate
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
REFERENCES
373
gyrus of the mouse hippocampus.91 The CRISPRi system modulated the excitatory–inhibitory balance of the dentate gyrus, altering learning and behavior in mice. Finally, multiplex CRISPRi abolished the expression of five genes important for neurotransmitter release in the brain, promoting the utility of in vivo CRISPRi to transcriptionally regulate mutant genes for neurological disease therapy. Gene editing with CRISPR–Cas allows for generally simple, precise gene modifications and regulation in the mammalian nervous system. The technology opens a new door in biomedical research for drug discovery, uncovering disease mechanisms, and personalized medicine and therapeutics. Both the recent successes of gene therapy in the clinic and in vivo CRISPR–Cas gene editing in animal models demonstrate tremendous potential for developing better models and for future treatment. Although further studies are needed to better understand in vivo off-target effects and to advance delivery systems for improved safety and reduced immunogenicity in the brain, CRISPR–Cas technology will continue to facilitate biomedical research and holds promise for application to a broad spectrum of neurological disorders.
References 1. Walters BJ, Azam AB, Gillon CJ, Josselyn SA, Zovkic IB. Advanced in vivo use of CRISPR/Cas9 and anti-sense DNA inhibition for gene manipulation in the brain. Front Genet. 2015;6:362. 2. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-1278. 3. Wright AV, Nunez JK, Doudna JA. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell. 2016;164(1-2):29-44. 4. Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709-1712. 5. Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565-1575. 6. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006;1:7. 7. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-823. 8. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. 9. Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659-662. 10. Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct. 2011;6:38. 11. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213). 12. Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827-832. 13. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-826. 14. Fishman-Lobell J, Rudin N, Haber JE. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992;12(3):1292-1303. 15. Auer TO, Del Bene F. CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods. 2014; 69(2):142-150. 16. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
374
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
17. Beumer KJ, Carroll D. Targeted genome engineering techniques in Drosophila. Methods. 2014;68(1):29-37. 18. Shin JW, Lee JM. The prospects of CRISPR-based genome engineering in the treatment of neurodegenerative disorders. Ther Adv Neurol Disord. 2018;11. 19. Maresca M, Lin VG, Guo N, Yang Y. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 2013;23(3):539-546. 20. Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPRCas system. Cell. 2015;163(3):759-771. 21. Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014; 159(2):440-455. 22. Liu Y, Ma S, Wang X, et al. Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9. Insect Biochem Mol Biol. 2014;49:35-42. 23. Zhou J, Shen B, Zhang W, et al. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol. 2014;46:49-55. 24. Shalem O, Sanjana NE, Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-87. 25. Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442-451. 26. Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23(10):1163-1171. 27. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013;10(10):977-979. 28. Perez-Pinera P, Kocak DD, Vockley CM, et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013;10(10):973-976. 29. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159(3):635-646. 30. Hu J, Lei Y, Wong WK, et al. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors. Nucleic Acids Res. 2014;42(7):4375-4390. 31. Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159(3):647-661. 32. Konermann S, Brigham MD, Trevino AE, et al. Genome-scale transcriptional activation by an engineered CRISPRCas9 complex. Nature. 2015;517(7536):583-588. 33. Ran FA, Cong L, Yan WX, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-191. 34. Staahl BT, Benekareddy M, Coulon-Bainier C, et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017;35(5):431-434. 35. Gao X, Tao Y, Lamas V, et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature. 2018;553(7687):217-221. 36. Carlson DF, Fahrenkrug SC, Hackett PB. Targeting DNA with fingers and TALENs. Mol Ther Nucleic Acids. 2012;1. 37. Ansai S, Kinoshita M. Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open. 2014;3(5):362-371. 38. Heidenreich M, Zhang F. Applications of CRISPR-Cas systems in neuroscience. Nat Rev Neurosci. 2016; 17(1):36-44. 39. Southwell AL, Skotte NH, Kordasiewicz HB, et al. In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides. Mol Ther. 2014;22(12):2093-2106. 40. Wu X, Kriz AJ, Sharp PA. Target specificity of the CRISPR-Cas9 system. Quant Biol. 2014;2(2):59-70. 41. Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R. TALEN or Cas9—rapid, efficient and specific choices for genome modifications. J Genet Genomics. 2013;40(6):281-289. 42. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279-284. 43. Kolli N, Lu M, Maiti P, Rossignol J, Dunbar GL. Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases. Neurochem Int. 2018;112:187-196. 44. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014;15(5):321-334. 45. Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380-1389.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
REFERENCES
375
46. Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genomewide off-target effects. Nature. 2016;529(7587):490-495. 47. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351(6268):84-88. 48. Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018;556(7699):57-63. 49. Rahdar M, McMahon MA, Prakash TP, Swayze EE, Bennett CF, Cleveland DW. Synthetic CRISPR RNA-Cas9guided genome editing in human cells. Proc Natl Acad Sci U S A. 2015;112(51):E7110-E7117. 50. Truong DJ, Kuhner K, Kuhn R, et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 2015;43(13):6450-6458. 51. Wright AV, Sternberg SH, Taylor DW, et al. Rational design of a split-Cas9 enzyme complex. Proc Natl Acad Sci U S A. 2015;112(10):2984-2989. 52. Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015;33(2):139-142. 53. Richter F, Fonfara I, Gelfert R, Nack J, Charpentier E, Moglich A. Switchable Cas9. Curr Opin Biotechnol. 2017;48:119-126. 54. Dai WJ, Zhu LY, Yan ZY, Xu Y, Wang QL, Lu XJ. CRISPR-Cas9 for in vivo gene therapy: promise and hurdles. Mol Ther Nucleic Acids. 2016;5:e349. 55. Anheim M, Tranchant C, Koenig M. The autosomal recessive cerebellar ataxias. N Engl J Med. 2012;366(7):636-646. 56. He X, Tan C, Wang F, et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homologydependent and independent DNA repair. Nucleic Acids Res. 2016;44(9):e85. 57. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature. 2017;551(7681):464-471. 58. Straub C, Granger AJ, Saulnier JL, Sabatini BL. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS One. 2014;9(8). 59. Swiech L, Heidenreich M, Banerjee A, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33(1):102-106. 60. McCarroll SA, Hyman SE. Progress in the genetics of polygenic brain disorders: significant new challenges for neurobiology. Neuron. 2013;80(3):578-587. 61. Tabebordbar M, Zhu K, Cheng JKW, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351(6271):407-411. 62. Nelson CE, Hakim CH, Ousterout DG, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403-407. 63. Long C, Amoasii L, Mireault AA, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351(6271):400-403. 64. Yang Y, Wang L, Bell P, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol. 2016;34(3):334-338. 65. Yin H, Song CQ, Dorkin JR, et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. 2016;34(3):328-333. 66. Gyorgy B, Loov C, Zaborowski MP, et al. CRISPR/Cas9 mediated disruption of the swedish APP allele as a therapeutic approach for early-onset Alzheimer’s disease. Mol Ther Nucleic Acids. 2018;11:429-440. 67. Poon A, Schmid B, Pires C, et al. Generation of a gene-corrected isogenic control hiPSC line derived from a familial Alzheimer’s disease patient carrying a L150P mutation in presenilin 1. Stem Cell Res. 2016;17(3):466-469. 68. Sundal C, Fujioka S, Uitti RJ, Wszolek ZK. Autosomal dominant Parkinson’s disease. Parkinsonism Relat Disord. 2012;18(suppl 1):S7-10. 69. Heman-Ackah SM, Bassett AR, Wood MJ. Precision modulation of neurodegenerative disease-related gene expression in human iPSC-derived neurons. Sci Rep. 2016;6. 70. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345(6201):1184-1188. 71. Ousterout DG, Kabadi AM, Thakore PI, Majoros WH, Reddy TE, Gersbach CA. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 2015;6:6244. 72. Cinesi C, Aeschbach L, Yang B, Dion V. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase. Nat Commun. 2016;7.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
376
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
73. An MC, Zhang N, Scott G, et al. Genetic correction of Huntington’s disease phenotypes in induced pluripotent stem cells. Cell Stem Cell. 2012;11(2):253-263. 74. Kolli N, Lu M, Maiti P, Rossignol J, Dunbar GL. CRISPR-Cas9 mediated gene-silencing of the mutant huntingtin gene in an in vitro model of Huntington’s disease. Int J Mol Sci. 2017;18(4). 75. Shin JW, Kim KH, Chao MJ, et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet. 2016;25(20):4566-4576. 76. Monteys AM, Ebanks SA, Keiser MS, Davidson BL. CRISPR/Cas9 editing of the mutant huntingtin allele in vitro and in vivo. Mol Ther. 2017;25(1):12-23. 77. Yang S, Chang R, Yang H, et al. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. J Clin Invest. 2017;127(7):2719-2724. 78. Merienne N, Vachey G, de Longprez L, et al. The self-inactivating KamiCas9 system for the editing of CNS disease genes. Cell Rep. 2017;20(12):2980-2991. 79. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72(2):245-256. 80. Renton AE, Majounie E, Waite A, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72(2):257-268. 81. Nimsanor N, Kitiyanant N, Poulsen U, et al. Generation of an isogenic, gene-corrected iPSC line from a symptomatic 57-year-old female patient with frontotemporal dementia caused by a P301L mutation in the microtubule associated protein tau (MAPT) gene. Stem Cell Res. 2016;17(3):556-559. 82. Wang L, Yi F, Fu L, et al. CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs. Protein Cell. 2017;8(5):365-378. 83. Gaj T, Ojala DS, Ekman FK, Byrne LC, Limsirichai P, Schaffer DV. In vivo genome editing improves motor function and extends survival in a mouse model of ALS. Sci Adv. 2017;3(12):eaar3952. 84. Park CY, Halevy T, Lee DR, et al. Reversion of FMR1 methylation and silencing by editing the triplet repeats in fragile X iPSC-derived neurons. Cell Rep. 2015;13(2):234-241. 85. Xie N, Gong H, Suhl JA, Chopra P, Wang T, Warren ST. Reactivation of FMR1 by CRISPR/Cas9-mediated deletion of the expanded CGG-repeat of the fragile X chromosome. PLoS One. 2016;11(10). 86. Liu XS, Wu H, Krzisch M, et al. Rescue of fragile X Syndrome neurons by DNA methylation editing of the FMR1 gene. Cell. 2018;172(5):979-992.e976. 87. Abudayyeh OO, Gootenberg JS, Essletzbichler P, et al. RNA targeting with CRISPR-Cas13. Nature. 2017; 550(7675):280-284. 88. Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. 2017; 358(6366):1019-1027. 89. Nelles DA, Fang MY, O’Connell MR, et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell. 2016;165(2):488-496. 90. Batra R, Nelles DA, Pirie E, et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell. 2017;170(5):899-912 e810. 91. Zheng Y, Shen W, Zhang J, et al. CRISPR interference-based specific and efficient gene inactivation in the brain. Nat Neurosci. 2018;21(3):447-454.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
18 CRISPR–Cas Gene Editing for Neurological Disease Bryan P. Simpson*,†, Beverly L. Davidson*,† *
The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, United States †The Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, United States
INTRODUCTION Many neurological diseases are caused by genetic mutations, which means they are amenable to various gene therapy approaches. Such approaches include gene augmentation to replace the mutant gene, or gene knockdown to inhibit expression of a dominantly acting disease gene. Therapeutic modalities also include going after the mutant protein, either by replacing it (e.g., enzyme replacement therapy) or by inhibiting it (e.g., introducing antibodies to remove toxic gene products). Recently, gene editing has been added to the toolbox of methodologies to reduce the disease gene burden. Among gene editing techniques is the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated nuclease (Cas) gene editing technology, referred to as CRISPR–Cas. This modality offers the flexibility to edit, replace, and modify specific loci in the genome with simplicity and ease in the laboratory, which makes it a useful tool to knockout and knock-in genes, to activate or silence gene expression, and to create model systems.1 The CRISPR–Cas system has been well characterized since its rise to fame and is now moving toward therapeutic development, having shown promise in disease models.2, 3 This chapter highlights the studies and findings supporting the feasibility and utility of therapeutic CRISPR–Cas gene editing as it applies to neurological disease.
CRISPR–Cas GENE EDITING IN MAMMALIAN SYSTEMS CRISPR is a bacterial adaptive immune system that targets foreign nucleic acid sequences (Fig. 1).4, 5 Bacteria use this system to defend against infections by incorporating short
Nervous System Drug Delivery https://doi.org/10.1016/B978-0-12-813997-4.00018-9
365
# 2019 Elsevier Inc. All rights reserved.
366
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
FIG. 1 The CRISPR–Cas adaptive immune system in prokaryotes. The clustered regularly interspaced short palindromic repeat (CRISPR) locus is a genomic region that provides adaptive immunity against viral infections in prokaryotes. When foreign nucleic acid is introduced into the host cell, the Cas operon expresses CRISPR-associated proteins (i.e., Cas) to select and integrate a protospacer sequence into the CRISPR array. The CRISPR array expresses pre-CRISPR RNA (crRNA) that is further processed into mature crRNA. The Cas nuclease protein complexes with the crRNA, which guides it to the sequence-specific target for cleavage.
fragments of the foreign DNA within the CRISPR region of its genome. The CRISPR region consists of short, repetitive, palindromic spacer sequences, and sequences encoding Cas proteins.6 When the foreign DNA fragments are expressed, they behave as guide RNAs (gRNAs) to direct the Cas nuclease to the invading target, which results in cleavage of the foreign agent’s DNA.1 The fusion of CRISPR RNA (crRNA) and the trans-activating CRISPR RNA (tracrRNA) into a chimeric single-guide RNA (sgRNA) allows for site-specific gene editing in eukaryote genomes (Fig. 2).7, 8 The CRISPR type II system with the Streptococcus pyogenes Cas9 (SpCas9) nuclease is the most commonly used system, and scientists take advantage of it by designing an sgRNA that targets a specific locus, and expressing the sgRNA along with Cas9 in vitro or in vivo.9 Cas9 associates with the sgRNA, and the complex is targeted to the desired DNA sequence.2, 10, 11 The Cas9-sgRNA complex binds to the protospacer adjacent motif (PAM) sequence located next to the sgRNA target sequence, and the two enzymatic endonuclease domains of Cas9 (i.e., RuvC and HNH) cleave both strands of DNA to generate a doublestrand break (DSB).7, 12, 13 The DSB can be repaired by one of the two DNA repair mechanisms: nonhomologous end joining (NHEJ) or homology-directed repair (HDR).14 NHEJ, the predominant repair pathway in mammalian cells, is prone to error and can result in insertion or deletion (i.e., indel) of nucleotides. Indels can create frameshift mutations and downstream premature stop codons, leading to inactivation of the target gene.2, 11
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
CRISPR–Cas GENE EDITING IN MAMMALIAN SYSTEMS
367
FIG. 2 CRISPR–Cas9 gene editing in eukaryotes. The sequence-specific Cas9-sgRNA complex binds the protospacer adjacent motif (PAM), the protospacer sequence of the sgRNA anneals; meanwhile, the catalytic nuclease domains (i.e., RuvC and HNH) cleave the target DNA sequence to create a double-strand break (DSB). The errorprone, nonhomologous end-joining (NHEJ) repair pathway can create insertion and deletion (i.e., indel) mutations that can result in a frameshift, premature stop codon, and, ultimately, gene knockout. Alternatively, the homologydirected repair pathway can modify or correct a gene by knock-in when a donor DNA template is provided.
In addition to gene knockout and inactivation, targeted gene insertion is possible by CRISPR–Cas9 HDR-mediated knock-in of donor DNA sequence templates,15-17 which can be harnessed to correct loss-of-functions mutations.18 HDR may be less efficient and somewhat limited in terminally differentiated postmitotic cells such as neurons.16 However, NHEJ in neurons can occur, allowing for knock-in editing strategies. The CRISPR-Cpf1 system with RNA-guided Cpf1, a nuclease like Cas9, generates DSBs with overhangs; this in turn promotes NHEJ in neurons.19-21 Finally, CRISPR–Cas9 multiplexing is possible when multiple sgRNAs are used to target different genes and pathways simultaneously, or when more than one guide is required to modify a gene.15, 22-24 The CRISPR–Cas9 system also allows for transcriptional regulation to modulate gene expression without permanently editing the genome (Fig. 3). For this, a mutated, dead Cas9 (dCas9) that lacks enzymatic endonuclease activity is used.25 The dCas9-sgRNA complex binds the targeted DNA sequence without creating a DSB, and is used to silence or activate gene expression (processes known as CRISPR interference [CRISPRi] or CRISPR activation
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
368
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
FIG. 3
dCas9-mediated gene manipulation. Catalytically inactive dead Cas9 (dCas9) can functionally bind target DNA to impart transcriptional downregulation. Additionally, when dCas9 is fused with different effector domains, it is possible to enhance gene repression, activate gene expression, target epigenetic modifications, or edit DNA bases.
[CRISPRa], respectively). This enhanced gene regulation is achieved with effector protein domains fused to dCas9 to recruit epigenetic modifying factors.25-29 For CRISPRi, the transcriptional repressor domain Kr€ uppel associated box (KRAB) is fused to dCas9; for CRISPRa, the transcriptional activator domain VP64 is fused to dCas9.25, 30 Alternatively, sgRNA can be modified with an MS2 coat protein fused to an effector domain for robust transcriptional modulation.31, 32 CRISPR–Cas9 is delivered to cells using standard viral and nonviral methods. Currently, adeno-associated viral (AAV) vectors are the most widely used delivery method for in vivo delivery of CRISPR–Cas9; however, AAV packaging size is restricted to 4.7 kb, so a dualAAV vector system is required. One vector delivers the SpCas9, and the second vector delivers the sgRNA(s). Alternatively, Staphylococcus aureus Cas9 (SaCas9), a smaller Cas9 orthologue, can fit into a single AAV vector along with the sgRNAs.33 Because viral delivery and constitutive expression of CRISPR–Cas9 may increase off-target effects and the risk of an immune response to the foreign bacterial protein, short-lived ribonucleoprotein (RNP) complexes of sgRNA-Cas9 are attractive alternatives for gene editing. RNP delivery has been shown to be effective after direct injections into mouse cortex, striatum, and hippocampus.34 Additionally, in vivo RNP delivery of an allele-specific sgRNA-Cas9 complex resulted in correction of hearing loss in a dominant deafness-associated allele in the Tmc1 Beethoven mouse model.35 These studies support the potential for RNP delivery in neurological diseases to overcome the safety concern of sustained expression from viral delivery systems, or the need for more than one vector. The CRISPR–Cas9 system provides advantages over alternative genome editing tools, such as zinc-finger nucleases and transcription activator-like effector nucleases.36 Most notably, CRISPR–Cas9 is easily adaptable to any gene and does not require extensive optimization, although editing frequency can vary depending on the target sequence and the sgRNAs.37 In addition, CRISPR–Cas9 overcomes the limitations of RNA-targeting
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
MOVING CRISPR–Cas GENE EDITING TOWARD THE CLINIC
369
strategies for gain-of-function mutations, such as RNA interference and antisense oligonucleotides. These latter approaches, if delivered via nonviral means, require repeated treatments, and both can be prone to high off-target effects and cytotoxicity.38, 39 One major drawback of the CRISPR–Cas9 system is off-target effects due to sgRNA sequence homology to other genomic sites.40, 41 Cas9 can tolerate three to five mismatches, depending on the distribution and number of mismatched nucleotides.2, 13, 30, 42 Designing sgRNAs with low GC content and minimal homology to off-target sites, reducing the size of the sgRNA, and limiting the amount of Cas9 protein are all ways to minimize off-targets.12, 42, 43 The nickase Cas9, a mutated Cas9 with only one functionally enzymatic endonuclease domain, also reduces offtargets because it cuts only one strand of the DNA. However, the nickase Cas9 does require two sgRNAs targeting opposite DNA strands to create a DSB for generation of the desired indel.44, 45 Engineering advances in the CRISPR–Cas9 system have focused on optimizing editing efficiency, specificity, and Cas9 regulation. On-target specificity was improved by engineering an enhanced Cas9 and a high-fidelity Cas9; both were associated with reduced offtargeting.46, 47 An evolved SpCas9 variant, xCas9, is associated with improved specificity, low off-target activity, and broader PAM compatibility, all of which facilitate broader applicability.48 Another approach involves a synthetic crRNA to replace the wild-type crRNA, which reduces off-targets and enhances cleavage activity.49 Additionally, split-Cas9 systems have been developed to control and regulate Cas9 expression. In this scenario, Cas9 is expressed by two expression systems that can be induced to dimerize, and has editing activity similar to that of wild-type SpCas9.50-52 Finally, expression regulated by small molecules, light, or temperature have also been designed to control Cas9 activity.53
MOVING CRISPR–Cas GENE EDITING TOWARD THE CLINIC Disease-modifying treatments for neurological diseases are currently lacking, with most drugs targeting symptom management. Early CRISPR–Cas9 genome editing studies were used to create model systems, but advances in gene delivery of CRISPR–Cas9 technology now support the clinical utility of this modality for genome editing.54 For dominantly inherited neurological diseases, CRISPR–Cas9 gene editing could inactivate or transcriptionally downregulate disease-causing gain-of-function mutations. Additionally, CRISPR–Cas9 repair of mutant genes could correct autosomal recessive loss-of-function mutations.55, 56 Even more exciting is the recent development of CRISPR–Cas9 single-base editing strategies for correcting pathogenic point mutations (Fig. 3).57 Not long after CRISPR–Cas9 gene editing was performed in eukaryotic cells, in vivo editing of genes was investigated in the mammalian brain. The initial CRISPR–Cas9 gene editing studies in postmitotic neurons and the mammalian brain support the technology as a viable therapeutic option.58, 59 The first proof-of-principle study of CRISPR–Cas9 gene knockdown in neurons was done in embryonic mice,58 and CRISPR–Cas9 knockout of the N-methyl-D-aspartate receptor (NMDAR) subunit protein GluN1 was done in mouse hippocampal pyramidal neurons after in utero electroporation delivery. NMDAR currents were abolished, demonstrating functional in vivo knockdown of Grin1. Additionally, the authors transfected the Cas9/gRNA complex into postmitotic neurons in hippocampal slice cultures
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
370
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
from P6-P8 rats and showed similar reductions in NMDAR-dependent currents. Although the study did not demonstrate in vivo editing of neurons after CRISPR–Cas9 delivery into the brain, it was nonetheless a tremendous step forward. In other work, Mecp2 promoter-driven Cas9 and an sgRNA targeting the Mecp2 gene demonstrated high editing efficiency with concomitant changes in learning and behavior.59 The CRISPR–Cas9 modifications were stable after AAV delivery to adult mice hippocampal neurons. CRISPR–Cas9 multiplexing can also target multiple genes, suggesting the potential for treating polygenic disease such as psychiatric disorders;60 in fact, Cas9 coupled with three sgRNAs targeting Dnmt3a, Dnmt1, and Dnmt3b in the hippocampus induced behavioral alterations, setting the stage for in vivo CRISPR–Cas9 interrogation of pathways to address the roles of pathways in complex behavior, with possible adaptation to clinical problems. CRISPR–Cas9 application to correct a monogenic disease was first presented by three groups studying Duchenne muscular dystrophy in mice models.61-63 They showed editing and improvement in phenotypes after delivery of the editing machinery. CRISPR–Cas9 gene therapy was also studied in the setting of hereditary liver disease.64, 65 These pioneering researchers opened the door for ongoing in vivo CRISPR–Cas9 gene editing research and the application of this research toward treating neurological diseases. Currently, CRISPR–Cas9 technology is geared toward correcting monogenic diseases in patient cell lines and animal models of Alzheimer disease (AD), Parkinson disease, amyotrophic lateral sclerosis (ALS), and the repeat expansion disorders, such as fragile X syndrome (FXS), Huntington disease (HD), the autosomal dominant spinocerebellar ataxias (SCAs), and spinal and bulbar muscular atrophy (SBMA). Alzheimer disease is a major cause of morbidity and mortality in elderly individuals. Mutations in the presenilin genes PSEN1 and PSEN2 and in the amyloid precursor protein (APP) gene result in amyloid-associated pathology that leads to early-onset familial AD, and variants of APOE are risk factors for late-onset AD.43 Gyorgy and colleagues disrupted a mutant variant of APP in human fibroblasts, providing the first experimental evidence of CRISPR– Cas9 gene editing for treatment of familial forms of dominant AD.66 PSEN1 mutations were corrected in AD-patient-induced pluripotent stem cells (iPSCs) by HDR-mediated CRISPR– Cas9 when combined with homologous repair templates.67 Mutations in LRRK2 and SNCA are causes of autosomal dominant Parkinson disease.68 CRISPRi targeting the transcription start site of SNCA significantly reduced expression levels in vitro.69 Additionally, studies using dCas9-KRAB repression and dCas9-VPR activation showed that SNCA expression could be modulated in human iPSC-derived neurons. Polyglutamine (polyQ) repeat expansion disorders such as HD, Kennedy disease, and SCAs are caused by toxic gain-of-function mutations. HD is a neurodegenerative disease caused by an expanded CAG trinucleotide repeat in the huntingtin gene (i.e., HTT), which results in a mutant HTT protein with an expanded polyQ repeat. Several CRISPR–Cas9 approaches have been investigated to reduce expression of mutant HTT. Similar to findings in mouse models of muscular dystrophy, CRISPR-mediated HDR has been suggested as an exon-skipping strategy to target polyQ repeats.70, 71 Another approach is to reduce the size of the expanded CAG repeat. Using the CRISPR–Cas9 D10A nickase resulted in CAG/ CTG repeat contraction in a reporter vector.72 The problem with this approach is that other repeat-containing genes in the genome may be targeted. Replacement of the expanded polyQ repeat with a normal polyQ repeat by homologous recombination in iPSCs from HD patients
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
MOVING CRISPR–Cas GENE EDITING TOWARD THE CLINIC
371
improved disease phenotypes, suggesting the potential for a similar HDR-mediated approach using CRISPR–Cas9.73 However, the efficiency of an HDR-mediated approach may not be sufficient to correct disease in humans. Alternatively, CRISPRi targeting the transcription start site of HTT resulted in reduction of HTT mRNA and protein expression levels.69 Meanwhile, CRISPR-NHEJ-mediated deletion reduced mutant HTT expression in mesenchymal stem cells from the YAC128 mouse model of HD.74 CRISPR–Cas9 targeted deletion or contraction of the HTT polyQ repeat might not restore normal protein function, and whether complete knockout of both the mutant and wild-type HTT alleles will be tolerated remains unknown. To overcome these problems, allele-specific editing of the mutant allele was demonstrated with CRISPR–Cas9 by using single-nucleotide polymorphisms (SNPs) found in PAMs of the mutant allele. In one approach, two allelespecific sgRNAs targeting SNPs within PAMs on the mutant allele created a large deletion including the promoter (i.e., TSS), and expanded CAG from the mutant allele in patient fibroblasts, iPSCs, and neural progenitor cells.75 Ablation of mutant HTT mRNA and protein demonstrated the efficacy of allele-specific CRISPR–Cas9 without targeting the normal allele. A similar approach identified SNPs within PAMs flanking exon-1 of HTT in heterozygosity between the normal and mutant alleles.76 Allele-specific deletion of HTT exon-1 resulted in targeted mutant HTT mRNA and protein knockdown in human HD fibroblasts. In vivo allelespecific editing in the BACHD transgenic mouse model reduced human mutant HTT expression to 40%, a level known to provide therapeutic benefit by RNA interference or antisense oligonucleotides, demonstrating CRISPR–Cas9 in vivo efficacy in an HD mouse model for the first time. A nonallele-specific approach with sgRNAs flanking the CAG repeat reduced human mutant HTT in striatal neurons and improved motor deficits in the HD140Q-knock-in mouse model of HD, which shows the behavioral effects of mutant HTT reduction by in vivo CRISPR–Cas9.77 To address constitutive Cas9 expression, an off-switch was engineered and tested in HD patient-derived neurons and in a mouse brain.78 The “KamiCas9” contains an sgRNA that can reduce not only the target, but also the nuclease. Although this may reduce immune responses to the bacterial nuclease, further testing is required to determine the extent to which this also may alleviate unintended editing. The expansion of GGGGCC hexanucleotide (G4C2) repeats in the C9orf72 gene cause familial ALS and frontotemporal dementia.79, 80 Mutihac and colleagues (cited in Ref 18) corrected an expanded G4C2 repeat with HDR-mediated CRISPR–Cas9 in iPSCs from an individual with a C9orf72 mutation, and ameliorated disease-associated phenotypes such as protein aggregation and stress granule formation in differentiated motor neurons.18 In other work, mutations in microtubule-associated protein tau were corrected in iPSCs derived from individuals with frontotemporal dementia.81 Autosomal dominant mutations in SOD1 and FUS cause familial ALS. Single-nucleotide mutations in SOD1 and FUS were corrected by HDR-mediated CRISPR–Cas9 in iPSCs from individuals with familial ALS.82 Systemic AAV9 delivery of SaCas9 and an sgRNA targeting exon 2 of human SOD1 reduced mutant SOD1 expression by more than 2.5-fold in the spinal cord of neonatal G93A-SOD1 model mice of ALS,83 improving motor function and slowing muscle atrophy. Furthermore, the mice showed a delay in disease onset, more surviving neurons at end stage, and increased survival.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
372
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
Fragile X syndrome is a neurodevelopmental disorder that causes learning disabilities and cognitive impairment. FXS is a trinucleotide repeat disorder caused by CGG repeat expansion in the 50 UTR of the fragile X mental retardation 1 (FMR1) gene. This causes hypermethylation of FMR1 with transcriptional silencing and reduced fragile X mental retardation protein (FMRP) expression. CRISPR–Cas9 and an sgRNA targeting upstream of the FMR1 CGG repeat in patient FXS iPSCs resulted in deletion of CGG repeats and reactivation of FMR1 expression.84 In another study in FXS iPSCs, Cas9 was expressed along with two sgRNAs flanking the CGG repeat, resulting in precise, efficient removal of the repeat and increased FMR1 expression.85 An alternative approach targeted the hypermethylation of FMR1 with dCas9-Tet for demethylation of the CGG expansion at the FMR1 promoter to reactivate expression of FMR1 in FXS iPSCs.86 The edited iPSCs that were subsequently differentiated into neurons had wild-type phenotypes, and maintained FMR1 expression after engraftment into the mouse brain. Notably, in vivo CRISPR–Cas9 editing and reactivation of FMR1 has not yet been shown in vivo. A related disorder, fragile X-associated tremor/ataxia (FXTAS), is caused by smaller repeat expansions in this locus, referred to as premutation of FMR1, and is a lateonset neurodegenerative disease caused by increased FMR1 mRNA toxicity and decreased FMRP expression. Based on the CRISPR–Cas9 studies in FXS iPSCs, similar strategies to edit and correct FMR1 in FXTAS are worth pursuing.
CRISPR–Cas RNA TARGETING A new generation of CRISPR–Cas proteins is making it possible to study RNA biology and to develop RNA-targeting therapeutics. Programmable RNA-guided Cas13, an RNase protein, efficiently reduces target mRNAs, and a catalytically inactive dCas13 edited single-bases in mammalian cells.87, 88 RNA-targeting CRISPR–Cas9 methods can reduce mRNAs associated with HD, C9orf72-linked ALS, FTXTAS, SBMA, the SCAs, and myotonic dystrophy types 1 and 2 (DM1 and DM2). An engineered RNA-targeting SpCas9 (RCas9), which can bind RNA, eliminated exogenous expression of CUG, CCUG, CAG, and GGGGCC repeat expansion RNAs in cells, and endogenous CUG and CCUG expansions in DM1 and DM2 patient cells.89, 90 Targeting expanded CAG RNAs reduced disease-associated polyQ protein, supporting the approach in HD, SBMA, and the SCAs. The advantage of RNA targeting is that no permanent off-target effects at the DNA level have been observed, which makes it a potentially safer strategy. However, repeated dosing or sustained expression from vector systems will be required, raising the specter of off-targeting or immune responses to the foreign Cas proteins. Studying RNA-targeting CRISPR systems in vivo will be the next step toward an RNA-directed therapy for neurological diseases.
CRISPR TRANSCRIPTIONAL REGULATION IN THE MAMMALIAN BRAIN Transcriptional regulation with dCas9 has been widely used in vitro to modulate gene expression. The first proof-of-principle use of in vivo CRISPRi in the mammalian brain was done with a lentivirus expressing dCas9-KRAB to knockdown Syt1 expression in the dentate
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
REFERENCES
373
gyrus of the mouse hippocampus.91 The CRISPRi system modulated the excitatory–inhibitory balance of the dentate gyrus, altering learning and behavior in mice. Finally, multiplex CRISPRi abolished the expression of five genes important for neurotransmitter release in the brain, promoting the utility of in vivo CRISPRi to transcriptionally regulate mutant genes for neurological disease therapy. Gene editing with CRISPR–Cas allows for generally simple, precise gene modifications and regulation in the mammalian nervous system. The technology opens a new door in biomedical research for drug discovery, uncovering disease mechanisms, and personalized medicine and therapeutics. Both the recent successes of gene therapy in the clinic and in vivo CRISPR–Cas gene editing in animal models demonstrate tremendous potential for developing better models and for future treatment. Although further studies are needed to better understand in vivo off-target effects and to advance delivery systems for improved safety and reduced immunogenicity in the brain, CRISPR–Cas technology will continue to facilitate biomedical research and holds promise for application to a broad spectrum of neurological disorders.
References 1. Walters BJ, Azam AB, Gillon CJ, Josselyn SA, Zovkic IB. Advanced in vivo use of CRISPR/Cas9 and anti-sense DNA inhibition for gene manipulation in the brain. Front Genet. 2015;6:362. 2. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262-1278. 3. Wright AV, Nunez JK, Doudna JA. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell. 2016;164(1-2):29-44. 4. Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709-1712. 5. Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565-1575. 6. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006;1:7. 7. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-823. 8. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. 9. Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659-662. 10. Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct. 2011;6:38. 11. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213). 12. Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827-832. 13. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-826. 14. Fishman-Lobell J, Rudin N, Haber JE. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992;12(3):1292-1303. 15. Auer TO, Del Bene F. CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods. 2014; 69(2):142-150. 16. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
374
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
17. Beumer KJ, Carroll D. Targeted genome engineering techniques in Drosophila. Methods. 2014;68(1):29-37. 18. Shin JW, Lee JM. The prospects of CRISPR-based genome engineering in the treatment of neurodegenerative disorders. Ther Adv Neurol Disord. 2018;11. 19. Maresca M, Lin VG, Guo N, Yang Y. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 2013;23(3):539-546. 20. Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPRCas system. Cell. 2015;163(3):759-771. 21. Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014; 159(2):440-455. 22. Liu Y, Ma S, Wang X, et al. Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9. Insect Biochem Mol Biol. 2014;49:35-42. 23. Zhou J, Shen B, Zhang W, et al. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol. 2014;46:49-55. 24. Shalem O, Sanjana NE, Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-87. 25. Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442-451. 26. Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23(10):1163-1171. 27. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013;10(10):977-979. 28. Perez-Pinera P, Kocak DD, Vockley CM, et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013;10(10):973-976. 29. Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159(3):635-646. 30. Hu J, Lei Y, Wong WK, et al. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors. Nucleic Acids Res. 2014;42(7):4375-4390. 31. Gilbert LA, Horlbeck MA, Adamson B, et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159(3):647-661. 32. Konermann S, Brigham MD, Trevino AE, et al. Genome-scale transcriptional activation by an engineered CRISPRCas9 complex. Nature. 2015;517(7536):583-588. 33. Ran FA, Cong L, Yan WX, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-191. 34. Staahl BT, Benekareddy M, Coulon-Bainier C, et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017;35(5):431-434. 35. Gao X, Tao Y, Lamas V, et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature. 2018;553(7687):217-221. 36. Carlson DF, Fahrenkrug SC, Hackett PB. Targeting DNA with fingers and TALENs. Mol Ther Nucleic Acids. 2012;1. 37. Ansai S, Kinoshita M. Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open. 2014;3(5):362-371. 38. Heidenreich M, Zhang F. Applications of CRISPR-Cas systems in neuroscience. Nat Rev Neurosci. 2016; 17(1):36-44. 39. Southwell AL, Skotte NH, Kordasiewicz HB, et al. In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides. Mol Ther. 2014;22(12):2093-2106. 40. Wu X, Kriz AJ, Sharp PA. Target specificity of the CRISPR-Cas9 system. Quant Biol. 2014;2(2):59-70. 41. Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R. TALEN or Cas9—rapid, efficient and specific choices for genome modifications. J Genet Genomics. 2013;40(6):281-289. 42. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279-284. 43. Kolli N, Lu M, Maiti P, Rossignol J, Dunbar GL. Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases. Neurochem Int. 2018;112:187-196. 44. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014;15(5):321-334. 45. Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380-1389.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
REFERENCES
375
46. Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genomewide off-target effects. Nature. 2016;529(7587):490-495. 47. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351(6268):84-88. 48. Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018;556(7699):57-63. 49. Rahdar M, McMahon MA, Prakash TP, Swayze EE, Bennett CF, Cleveland DW. Synthetic CRISPR RNA-Cas9guided genome editing in human cells. Proc Natl Acad Sci U S A. 2015;112(51):E7110-E7117. 50. Truong DJ, Kuhner K, Kuhn R, et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 2015;43(13):6450-6458. 51. Wright AV, Sternberg SH, Taylor DW, et al. Rational design of a split-Cas9 enzyme complex. Proc Natl Acad Sci U S A. 2015;112(10):2984-2989. 52. Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotechnol. 2015;33(2):139-142. 53. Richter F, Fonfara I, Gelfert R, Nack J, Charpentier E, Moglich A. Switchable Cas9. Curr Opin Biotechnol. 2017;48:119-126. 54. Dai WJ, Zhu LY, Yan ZY, Xu Y, Wang QL, Lu XJ. CRISPR-Cas9 for in vivo gene therapy: promise and hurdles. Mol Ther Nucleic Acids. 2016;5:e349. 55. Anheim M, Tranchant C, Koenig M. The autosomal recessive cerebellar ataxias. N Engl J Med. 2012;366(7):636-646. 56. He X, Tan C, Wang F, et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homologydependent and independent DNA repair. Nucleic Acids Res. 2016;44(9):e85. 57. Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature. 2017;551(7681):464-471. 58. Straub C, Granger AJ, Saulnier JL, Sabatini BL. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS One. 2014;9(8). 59. Swiech L, Heidenreich M, Banerjee A, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33(1):102-106. 60. McCarroll SA, Hyman SE. Progress in the genetics of polygenic brain disorders: significant new challenges for neurobiology. Neuron. 2013;80(3):578-587. 61. Tabebordbar M, Zhu K, Cheng JKW, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351(6271):407-411. 62. Nelson CE, Hakim CH, Ousterout DG, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403-407. 63. Long C, Amoasii L, Mireault AA, et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351(6271):400-403. 64. Yang Y, Wang L, Bell P, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol. 2016;34(3):334-338. 65. Yin H, Song CQ, Dorkin JR, et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. 2016;34(3):328-333. 66. Gyorgy B, Loov C, Zaborowski MP, et al. CRISPR/Cas9 mediated disruption of the swedish APP allele as a therapeutic approach for early-onset Alzheimer’s disease. Mol Ther Nucleic Acids. 2018;11:429-440. 67. Poon A, Schmid B, Pires C, et al. Generation of a gene-corrected isogenic control hiPSC line derived from a familial Alzheimer’s disease patient carrying a L150P mutation in presenilin 1. Stem Cell Res. 2016;17(3):466-469. 68. Sundal C, Fujioka S, Uitti RJ, Wszolek ZK. Autosomal dominant Parkinson’s disease. Parkinsonism Relat Disord. 2012;18(suppl 1):S7-10. 69. Heman-Ackah SM, Bassett AR, Wood MJ. Precision modulation of neurodegenerative disease-related gene expression in human iPSC-derived neurons. Sci Rep. 2016;6. 70. Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345(6201):1184-1188. 71. Ousterout DG, Kabadi AM, Thakore PI, Majoros WH, Reddy TE, Gersbach CA. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 2015;6:6244. 72. Cinesi C, Aeschbach L, Yang B, Dion V. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase. Nat Commun. 2016;7.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES
376
18. CRISPR–Cas GENE EDITING FOR NEUROLOGICAL DISEASE
73. An MC, Zhang N, Scott G, et al. Genetic correction of Huntington’s disease phenotypes in induced pluripotent stem cells. Cell Stem Cell. 2012;11(2):253-263. 74. Kolli N, Lu M, Maiti P, Rossignol J, Dunbar GL. CRISPR-Cas9 mediated gene-silencing of the mutant huntingtin gene in an in vitro model of Huntington’s disease. Int J Mol Sci. 2017;18(4). 75. Shin JW, Kim KH, Chao MJ, et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet. 2016;25(20):4566-4576. 76. Monteys AM, Ebanks SA, Keiser MS, Davidson BL. CRISPR/Cas9 editing of the mutant huntingtin allele in vitro and in vivo. Mol Ther. 2017;25(1):12-23. 77. Yang S, Chang R, Yang H, et al. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. J Clin Invest. 2017;127(7):2719-2724. 78. Merienne N, Vachey G, de Longprez L, et al. The self-inactivating KamiCas9 system for the editing of CNS disease genes. Cell Rep. 2017;20(12):2980-2991. 79. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011;72(2):245-256. 80. Renton AE, Majounie E, Waite A, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011;72(2):257-268. 81. Nimsanor N, Kitiyanant N, Poulsen U, et al. Generation of an isogenic, gene-corrected iPSC line from a symptomatic 57-year-old female patient with frontotemporal dementia caused by a P301L mutation in the microtubule associated protein tau (MAPT) gene. Stem Cell Res. 2016;17(3):556-559. 82. Wang L, Yi F, Fu L, et al. CRISPR/Cas9-mediated targeted gene correction in amyotrophic lateral sclerosis patient iPSCs. Protein Cell. 2017;8(5):365-378. 83. Gaj T, Ojala DS, Ekman FK, Byrne LC, Limsirichai P, Schaffer DV. In vivo genome editing improves motor function and extends survival in a mouse model of ALS. Sci Adv. 2017;3(12):eaar3952. 84. Park CY, Halevy T, Lee DR, et al. Reversion of FMR1 methylation and silencing by editing the triplet repeats in fragile X iPSC-derived neurons. Cell Rep. 2015;13(2):234-241. 85. Xie N, Gong H, Suhl JA, Chopra P, Wang T, Warren ST. Reactivation of FMR1 by CRISPR/Cas9-mediated deletion of the expanded CGG-repeat of the fragile X chromosome. PLoS One. 2016;11(10). 86. Liu XS, Wu H, Krzisch M, et al. Rescue of fragile X Syndrome neurons by DNA methylation editing of the FMR1 gene. Cell. 2018;172(5):979-992.e976. 87. Abudayyeh OO, Gootenberg JS, Essletzbichler P, et al. RNA targeting with CRISPR-Cas13. Nature. 2017; 550(7675):280-284. 88. Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. 2017; 358(6366):1019-1027. 89. Nelles DA, Fang MY, O’Connell MR, et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell. 2016;165(2):488-496. 90. Batra R, Nelles DA, Pirie E, et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell. 2017;170(5):899-912 e810. 91. Zheng Y, Shen W, Zhang J, et al. CRISPR interference-based specific and efficient gene inactivation in the brain. Nat Neurosci. 2018;21(3):447-454.
II. NERVOUS SYSTEM DRUG DELIVERY TECHNIQUES