Journal Pre-proof

CRISPR/Cas9: A preclinical and clinical perspective for the treatment of human

diseases

Garima Sharma, Ashish Ranjan Sharma, Manojit Bhattacharya, Sang-Soo Lee,

Chiranjib Chakraborty
PII: S1525-0016(20)30485-8
DOI: https://doi.org/10.1016/j.ymthe.2020.09.028
Reference: YMTHE 5280

To appear in: Molecular Therapy

Please cite this article as: Sharma G, Sharma AR, Bhattacharya M, Lee S-S, Chakraborty C, CRISPR/
Cas9: A preclinical and clinical perspective for the treatment of human diseases, Molecular Therapy
(2020), doi: https://doi.org/10.1016/j.ymthe.2020.09.028.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2020
Jo
ur
na
lP
re
-p
ro
of
 1 CRISPR/Cas9: A preclinical and clinical perspective for the treatment of human

2 diseases

3 Garima Sharma1#, Ashish Ranjan Sharma2#, Manojit Bhattacharya2, Sang-Soo Lee2*,

4 Chiranjib Chakraborty2,3*,

5
1
6 Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon

7 National University, Chuncheon, 24341, Republic of Korea

2
8 Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred

of
9 Heart Hospital, Chuncheon, Gangwon-Do, 24252, Republic of Korea.

ro
3
10 Department of Biotechnology, School of Life Science and Biotechnology, Adamas

11
-p
University, Barasat-Barrackpore Rd, Kolkata, West Bengal 700126, India.
re
12
lP

13 #Both authors contributed equally.

na

14 *Correspondence to:
ur

15 Prof. Sang-Soo Lee MD, PhD

Institute for Skeletal Aging & Orthopedic Surgery, Hallym University Hospital-College of
Jo

16

17 Medicine, Chuncheon-si, Gangwon-do 200-704, Republic of Korea

18 E-mail: [email protected], Tel: +82-33-240-5197, Fax: +82-33-252-0177

19

20 Prof. Chiranjib Chakraborty, PhD

21 Department of Biotechnology, School of Life Science and Biotechnology, Adamas

22 University, Barasat-Barrackpore Rd, Jagannathpur, Kolkata, West Bengal 700126, India.

23 E-mail: [email protected] Tel: +91-9871608125

24

25

1
26 Abstract:

27 At present, the idea of genome modification has revolutionized the modern therapeutic

28 research era. Genome modification studies have travelled a long way from gene

29 modifications in primary cells to genetic modifications in animal. The targeted genetic

30 modification may result in the modulation (i.e. either upregulation or downregulation) of the

31 predefined gene expression. Clustered regularly interspaced short palindromic repeats

32 (CRISPR) associated nuclease 9 (CRISPR/Cas9) is a promising genome editing tool that has

33 therapeutic potential against incurable genetic disorders by modifying their DNA sequences.

of
34 In comparison with other genome editing techniques, CRISPR/Cas9 is simple, efficient and

ro
35 very specific. This enabled CRISPR/Cas9 genome editing technology to enter into the

36
-p
clinical trials against cancer. Besides therapeutic potential, CRISPR/Cas9 tool can also be
re
37 applied to generate genetically inhibited animal models for drug discovery and development.
lP

38 This comprehensive review paper discussed the origin of CRISPR/Cas9 systems and its
na

39 therapeutic potential against various genetic disorders includes cancer, allergy,

40 immunological disorders, duchenne muscular dystrophy, cardiovascular disorders,

ur

41 neurological disorders, liver-related disorders, cystic fibrosis, blood-related disorders, eye-

Jo

42 related disorders and viral infection. Finally, we talk about the different challenges, safety

43 concerns and the strategies that can be applied to overcome the obstacles during

44 CRISPR/Cas9-mediated therapeutic approach.

45

46 Keywords: Human diseases, CRISPR/Cas9, genome editing, therapeutics, drug development

47

2
48 1. Introduction

49 Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR

50 associated protein 9 (Cas9) is a novel and competent RNA-guided endonuclease-based

51 genome editing technique1, that is adapted from naturally occurring bacterial immune system.

52 CRISPR/Cas9 technique is easily scalable and very cost-effective, thus, it can be applied for a

53 wide range of directed genome-editing.2-4 After first report of CRISPR in 1987 the

54 technology have evolved step by step (Fig. 1). Now a days, CRISPR/Cas9 technology-

55 mediated genetic experiments can be performed on a wide range of models such as plants,

of
56 yeast, Caenorhabditis elegans, Drosophila, zebrafish, mouse, and humans.5-11 More than

ro
57 2000 publications have been recorded from ‘Pubmed’ from the last two years. This increasing

58
-p
research trend was portrayed as the ‘CRISPR craze’.12
re
59 CRISPR/Cas9 technology utilizes single guide RNA (sgRNA) sequence and Cas9
lP

60 endonuclease. sgRNA is a combination of CRISPR RNA (crRNA) and trans-activating

na

61 crRNA (tracrRNA), that identify and attach the sgRNA:Cas9 ribonucleoprotein complex to

62 the target DNA.13 After DNA targeting, Cas9 generates double-stranded breaks (DSBs) at
ur

63 the site of target DNA.14 These DSBs are further repaired by insertions, deletions, additions
Jo

64 or inversions. The DNA repair mechanism can be performed by either host’s natural repair

65 machinery or by using, customized DNA sequences.15

66 The highlights of the CRISPR/Cas9 technology is its ability to treat various human

67 diseases via genome editing (Fig. 2).16 Accumulating evidence suggests successful genome

68 editing in mammalian cells via CRISPR/Cas9 technology.17,18 CRISPR/Cas9 technology has

69 now entered under clinical trials in USA committee (NIH Recombinant DNA Advisory

70 Committee) that has permitted the CRISPR technology to assist cancer therapies relying on

71 enlisting a patient’s T cells at the National Institutes of Health (NIH), USA19 In addition, an

72 analogous type of gene therapy clinical trials received ethical approval in China.20 These

3
73 clinical trials might establish an efficient therapeutic genome editing system against

74 hereditary or non-hereditary genetic disorders in humans.

75 Although CRISPR/Cas9 tool has the therapeutic potential, unexpected outcomes has

76 challenged the development of more simple and specific gene editing methodologies.21 In this

77 paper, we discuss the preclinical studies on the mechanism of action of CRISPR/Cas9

78 systems against various diseases, such as cancer, allergy and immunological disorders. We

79 also discuss about the clinical advances, challenges, safety concerns and the strategies to

80 develop efficient CRISPR/Cas9-based gene editing technology.

of
81

ro
82 2. Advantages of CRISPR/Cas9 and other genome editing tools

83
-p
Genome-editing technology has gained attention as a therapy against human diseases.
re
84 However, its success has been previously challenged by the unpredictable outcomes. In the
lP

85 early gene therapy trials five children, out of 20 participants from different studies, suffering
na

86 from severe combined immunodeficiency (SCID) X-1 condition developed T-cell leukemia

87 due to non-specific insertion of correcting gene near to tumor promoting genes that resulted
ur

88 in transcriptional activation.22-26 In another trial, 18-years old male with ornithine

Jo

89 transcarbamylase (OTC), developed lethal immune response induced by viral vector.27 Both

90 the misfortunes were associated with poor or uncontrolled therapeutic delivery methods.24

91 Lately, more advanced gene therapy technologies, such as zinc finger nucleases

92 (ZFNs)28, transcription activator-like effector nucleases (TALENs) 29

, and CRISPR/Cas9,30

93 were developed which were capable of site-specific gene editing. ZFNs is a combination of

94 non-specific FokI cleavage domain and zinc finger proteins (ZFPs). In eukaryotes, ZFPs are

95 associated with protein-protein interaction and regulation of DNA transcription.31 For gene

96 editing, paired ZFNs, each for downstream and upstream of target site, are used to generate

97 DSBs.32 ZFNs, however, can only recognize nucleotide triplets in the DNA, thus restricting

4
 98 the number of site selection due to limited binding sites for zinc finger proteins.33 Various

99 interventional clinical trials based on ZFNs-mediated gene editing are currently ongoing, e.g.

100 for the treatment of Hunter’s syndrome,34 to cure human immunodeficiency virus (HIV)

101 infection,35 etc. Similar to ZFNs, TALENs is a complex of transcription activator-like

102 effector (TALEs) and FokI endocuclease.29 TALEs are the amino acid sequences that flanks

103 DNA binding site. TALENs has benefit over ZFNs, as it can recognize single nucleotide and

104 is more specific than ZFNs.36 Still, packaging and delivery of TALENs might be a challenge

105 because of its large size than ZFNs.24

of
106 CRISPR/Cas9 is latest gene editing technique with various advantages over ZFNs and

ro
107 TALENs. Firstly, CRISPR/Cas9 is more cost-effective than other gene editing techniques.37

108
-p
Secondly, the target specificity of CRISPR/Cas9 depends on the ribonucleotide complex,
re
109 which is probably more specific to protein-based DNA bindings in ZFNs and TALENs.
lP

110 Moreover, it is easy to design sgRNA for a wide range of target DNA. In addition, various
na

111 modifications can be performed in CRISPR/Cas9 cargo system, i.e., plasmid DNA encoding

112 sgRNA and Cas9, combination of sgRNA and Cas9 mRNA and combination of sgRNA and
ur

113 Cas9 protein. Other advantages of CRISPR/Cas9 includes possibility of direct genome
Jo

114 modification in the embryo and introduction of more than one mutations at the same time.38

115 In addition, base edition [i.e., conversion of cytidine to uracil (C → T) or Guanine to Adenine

116 (G → A)] that is mediated by the binding of CRISPR/Cas9 with cytidine deaminase is also

117 possible.39 These advancements indicate the possibility of safe and successful clinical

118 application of CRISPR/Cas9 technology in future.

119

120 3. Preclinical studies of CRISPR/Cas9

121 CRISPR/Cas9 research has applied for the treatment of different human diseases

122 (table1) which are discussed as followed.

5
123 3.1. Cancer

124 Cancer is among the most prevalent lethal diseases that can be illustrated by the

125 accumulation of epigenetic modifications in the genome.40, 41 The drawback of conventional

126 chemotherapy is the lack of specific targeting, and resistance to chemotherapeutic drugs.42

127 Therefore, there is a need to identify novel molecular targets that may facilitate cancer

128 treatment. It has been proposed that the mutated oncogenes and tumor suppressor genes in

129 cancer cells might function as smart therapeutic targets, suggesting that the modulation of of

130 tumor suppressor genes can induce apoptosis in tumor cells.43,44 Liu et al. (2014) observed

of
131 effective inhibition of bladder cancer cell proliferation, reduced cell motility and induction of

ro
132 apoptosis via CRISPR/Cas9-mediated regulation of tumor suppressor genes, i.e. hBax, E-

133 cadherin and p21.45

-p
re
134 It has been reported that epigenetic regulators are often mutated in myeloid
lP

135 malignancies.46 CRISPR/Cas9-technology corrected additional sex combs like 1 (ASXL1)

na

136 gene and re-established ASXL1 protein expression that significantly decrease leukemia cell

137 growth in mouse xenografts.47 CRISPR/Cas9 system was also used to delete myeloid cell
ur

138 leukemia-1 (MCL-1) gene, a member of the emerging B-cell lymphoma 2 (BCL2) gene
Jo

139 family, in human Burkitt lymphoma (BL) cells for the induction of apoptosis in the BL

140 cells,48 suggesting that MCL1 gene can be a novel target for cancer treatment as it plays a

141 role in cell differentiation, proliferation, and tumorigenesis.49

142 Cyclin dependent kinases (CDKs) are critical regulator for cell cycle. Therefore,

143 dysregulated activation of CDKs might lead to tumorigenesis.50, 51 CRISPR/Cas9 technology

144 can silence the CDK11 gene for the treatment of osteosarcoma,52 and CDK7 gene for the

145 treatment of triple-negative breast cancer cells.53 This indicates that CDKs can be novel

146 targets for cancer treatment.

6
147 Resistance to chemotherapeutic drugs is a common disadvantage of chemotherapy.

148 Multidrug resistance gene 1 (MDR1) gene encodes for membrane efflux pump P-

149 glycoprotein. The overexpression of MDR1 facilitates the efflux of anti-cancer drugs from

150 the cells that results in chemotherapeutic drug resistance. CRISPR-Cas9-mediated

151 knockdown of MDR1 in osteosarcoma cell lines restored the sensitivity towards

152 chemotherapeutic drug.54 Likewise, the possible management of drug resistance acquired by

153 the secondary mutation in exon 20 at position 790 (T790M) in epidermal growth factor

154 receptor (EGFR) can also be an effective strategy to cure lung cancer.55

of
155 SHC SH2-domain binding protein 1 (SHCBP1) is a member of collagen homolog

ro
156 family that is critical for the regulation of cell proliferation. As the over expression of

157
-p
SHCBP1 gene is reported in several diseases especially cancer, it may be a potential
re
158 therapeutic target as well as suitable diagnostic biomarker for cancer.56 It has been
lP

159 demonstrated that the CRISPR/Cas9-mediated knockout of SHCBP1 gene might inhibit
na

160 cancer cell proliferation and induce apoptosis in breast cancer cells.56

161 Kelch-like (KLHL) gene family encodes a group of proteins that are related with the
ur

162 several human diseases, along with the cancer.57 It was observed that CRISPR/Cas9
Jo

163 mediated knockout of kelch domain containing 4 (KLHDC4) gene in a nasopharyngeal

164 carcinoma cell line considerably inhibited cancer cell migration and growth, and induced

165 apoptosis in both in vitro and in vivo models.58

166 In addition to therapeutic domain, CRISPR/Cas9-mediated modification of genes can

167 also be used to develop mutant cancer model in mice opening new opportunities to generate

168 mutant in various species and in almost any genetic background to accelerate in vivo

169 studies.59, 60 Platt et al. (2014) developed lung adenocarcinoma mice by knockout of three

170 significant genes [i.e., tumor protein (p53), serine/threonine kinase 11 (STK11) or Lkb1, and

171 kirsten rat sarcoma 2 viral oncogene homolog (KrasG12D)].10 In 2015, Chen et al. developed

7
172 in vivo loss-of-function Cas9 screening (Genome-wide CRISPR screen) for tumor metastasis

173 and growth study.61 CRISPR/Cas9 technology was also used to initiate multiple gene

174 mutations to develop human colonic epithelium in the colorectal cancer model.62

175 CRISPR/Cas9-mediated knockout of single gene, i.e. patched 1 (Ptch1), or multiple genes,

176 i.e., transformation-related protein 53 (TRP53), Neurofibromin 1 (Nf1), and phosphatase and

177 tensin homolog (PTEN), in the brain resulted in the progression of glioblastoma and

178 medulloblastoma in mouse.63 CRISPR/Cas9 technology was also used to generate acute

179 myeloid leukemia (AML) mouse model with combinatorial genetic lesions system by

of
180 inducing multiple mutations in the genes of epigenetic modifiers, cytokine signaling and

ro
181 transcription factors in the hematopoietic stem cells of mouse.64

182
-p
Cancer immunotherapy is among the four major line of treatments along with surgery,
re
183 chemotherapy and radiotherapy. Clinical trials on gene editing-based immunotherapies has
lP

184 been focusing on chimeric antigen receptor (CAR) T cell therapy65-67, and genetically
na

185 modified T-cell receptor (TCR) therapy. In recent years, Kymriah of Novartis and Yescarta

186 of KITE Pharma received USFDA approval on their CAR-T therapy products.68, 69 CAR, a
ur

187 synthetic receptor, acts as a gene insert that contains a transmembrane domain, a hinge
Jo

188 segment, an antibody-derived extracellular specific target protein binding domain and T-cell

189 activating intracellular signaling unit.70 CARs are inserted into the autologous T-cells that

190 are collected from the patients, resulting in the expression of CARs on the surface of T-cell.

191 When these T-cells constructs are introduced again into the patient, they multiply and binds

192 to the target protein and eliminates tumor cells. CAR-T therapy showed 80-100% remission

193 in patients with replapsed or refractory B-cell acute lymphocytic leukemia (ALL).65,71

194 However, cytokine releases during the therapy may result in various manegeable side effects.

195 While CARs recognize surface antigens, TCRs recognize intracellular proteins presented on

196 major histocompatibility complex I (MHCI). Moreover, TCR therapy is preferred over CAR-

8
197 T therapy due to low incidences cytokine release syndrome.72 Latest clinical trials are

198 working on the CRISPR/Cas9-based immunotherapy against various types of cancer, which

199 are detailed later in this review.

200

201 3.2. Allergy and immunological disorders

202 The therapeutic role of CRISPR/Cas9 genome editing for allergic and immunological

203 conditions has also been reported.73 It was observed that the expression of a cell surface

204 glycoprotein MUC18 or CD146 is increased in the alveolar macrophages of bacterial or viral

of
205 infections-mediated chronic obstructive pulmonary disease (COPD) or asthma.74 Knockout

ro
206 of MUC18 gene reduced the level of IL-8, a pro-inflammatory chemokine, in human primary

207
-p
nasal airway epithelial cells (AECs) that were stimulated by microbial infection mimicking
re
208 toll-like receptor (TLR) agonists (i.e, TLR-2, 3 and 4).75
lP

209 It was noted that targeting receptors genes, such as programmed death-1 (PD-1), can
na

210 stimulate immune responses. PD-1 is a T-cell surface protein that is associated with T-cell

211 activation. CRISPR/Cas9-mediated disruption of PD-1 receptor gene in human primary T

ur

212 cells isolated from cancer patients resulted in upregulated IFN-γ production and enhanced
Jo

213 cytotoxicity,76 suggesting checkpoint inhibitors as novel target for cancer treatment.

214 X-linked hyper immunoglobulin M syndrome is an immune deficiency disorder

215 identified by defective CD40/CD40L signaling via dysregulated class switch recombination

216 and somatic hypermutation in B-cells.77 It has been observed that CRISPR/Cas9 gene editing

217 technique can correct mutations in CD40 ligand.78 Cheong et al. (2016) edited the mouse and

218 human immunoglobulin (Ig) genes to obtain class switching of IgH which might assist in the

219 study of B cells (both normal and lymphoma) biology.79

220 JAK3 (Janus family kinase) is a protein tyrosine kinase which regulates various

221 pathogenic processes in allergic asthma.80 The deficiency in JAK3 is related to the reduced

9
222 number of circulating natural killer (NK) cells and T cells, and with normal numbers of

223 inadequately functioning B cells.81 It has been noted that the mutations in the JAK3 gene can

224 cause SCID. CRISPR/Cas9-mediated correction in human JAK3 gene restored the

225 differentiation potential of T cells progenitors that are capable of producing T cell/NK cells.81

226 This suggests that CRISPR/Cas9 technique can reprogram the cells for the prevention of

227 various allergic conditions.

228

229 3.3. Duchenne muscular dystrophy (DMD)

of
230 DMD is an X-linked disorder and is characterized by proximal muscle weakness

ro
231 caused by small mutations in the DMD gene that leads to the absence of dystrophinprotein. A

232
-p
number of knockout mouse models were produced to imitate the human DMD phenotype by
re
233 genetic inhibition of dystrophin and orutrophin/α7β1-integrin genes.82-84 It was demonstrated
lP

234 that genome editing can be used to restore the DMD gene mutation and correct the DMD
na

235 disorder.85-88 CRISPR/Cas9 technology has provided a platform for the treatment of DMD.1,
89
236 CRISPR/Cas9-mediated gene editing method was used to correct the DNA of an entire
ur

237 region containing CTG/CAG repeats suggesting a new therapeutic opportunity against
Jo

238 DMD.90 Moreover, gold nanoparticles were also used as delivery vehicle for Cas9

239 ribonucleoprotein and donor DNA to correct the DMD gene in mice with minimal off-target

240 DNA damage.91 Recently, Zhang et al. suggested that self-complementary adeno-associated

241 virus (ScAAV) delivery system can substantially improve the efficiency of CRISPR/Cas9-

242 mediated DMD gene correction.92

243

244 3.4. Cardiovascular disorders

245 Proprotein convertase subtilisin/kexin type 9 (PCSK9) gene has an important role in

246 the regulation of cholesterol homeostasis.93 The gain-of-function mutation of PCSK9 gene

10
247 can result in hypercholesterolemia and associated artherosclerosis.94 Jiang et al. (2017)

248 reported CRISPR/Cas9-mediated therapeutic targeting of PCSK9 gene in mice.95 In addition,

249 CRISPR/Cas9 genome editing tool was also reported to disrupt low-density lipoprotein

250 receptor (Ldlr) gene and overexpress PCSK9 gene in adult mice for atherosclerosis

251 research.96 Tessadori et al. (2018) applied CRISPR/Cas9-based genome editing tool in

252 zebrafish model to correct the human genetic cardiovascular disorders.97 These evidences

253 suggest the use of CRISPR/Cas9 as a potential genome editing tool against CVDs, especially

254 conditions associated with hereditary lipid disorders.

of
255

ro
256 3.5. Neurological disorders

257
-p
Huntington disease (HD) is an autosomal inherited neurological disorder that is
re
258 caused by the extension of CAG repeats in exon 1 of the huntington (HTT) gene.98
lP

259 CRISPR/Cas9 nucleotide editing tool was effectively applied to selectively suppress the HTT
na

260 gene in mouse model.99 Other studies also supported the use of CRISPR/Cas9 genome editing

261 system against HD condition.100, 101

ur

262 Alzheimer’s disease (AD), a neurodegenerative condition, leads to progressive

Jo

263 memory loss. Mutations in the presenilin 1 (PSEN1) and PSEN2 genes are reported in the

264 familial AD condition.102 PSEN1 is a catalytic subunit of γ-secretase, a protease enzyme that

265 cleaves amyloid precursor protein (APP) generating amyloid-β (Aβ). A79V mutation in

266 PSEN1 gene increases the Aβ42/Aβ40 ratio by decreasing Aβ40. Studies reported the

267 correction of A79V and L150P mutations in PSEN1 in induced pluripotent stem cell (iPSCs)

268 line derived from AD patient.103, 104 CRISPR/Cas9 nucleotide editing system was used to edit

269 the point mutation “T” with wild-type “C” nucleotide in A79V-hiPSC line. Moreover,

270 mutation in APP gene increases β-secretase cleavage of APP resulting in abnormally high Aβ

271 levels in brain. CRISPR/Cas9 was also used to correct APP allele and thereby decreasing Aβ

11
272 pathogenesis.105 CRISPR/Cas9-based genome editing against AD has been reviewed in detail

273 by Rohn et al. (2018).106 Therefore, CRISPR/Cas9 gene editing technology can be used as an

274 efficient strategy against genetically-induced neurological disorders.89

275

276 3.6. Metabolic disorders

277 Metabolic liver disease (MLD) is caused by the defect of a transporter protein that

278 results into abnormal metabolism of carbohydrate, protein and fat. Recently, Villiger et al.

279 (2018) corrected mutations in phenylalanine hydroxylase Pahenu2 gene to treat

of
280 phenylketonuria (PKU), an autosomal recessive liver disease, using CRISPR/Cas9 editor

ro
281 systems in mice.107 In another study, computationally designed hepatocyte-specific

282
-p
CRISPR/Cas9 was used to target murine factor IX (F9) gene against MLD-related
re
283 condition.108 Yang et al. (2016) worked on CRISPR/Cas9-mediated gene editing based
lP

284 correction of X-linked deficiency in ornithine transcarbamylase (OTC) to treat urea cycle
na

285 disorder in an infant mice model.109

ur

286 Hereditary tyrosinemia (HT) is an autosomal recessive inherited disease which is

287 associated with a deficiency of the enzyme fumaryl acetoacetate hydrolase due to mutations
Jo

288 in the Fah gene.110, 111 HT type I (HTI) causes severe hepatic disorders, such as cirrhosis, liver

289 failure, and hepatic cancer, due to toxin accumulation. The CRISPR technology-based

290 therapeutic strategy to correct HT was one of the first known studies in mice demonstrating

291 delivery of CRISPR/Cas9 system components to adult mammalian organs.112 In this study

292 reconstitution of a disease-causing mutation in Fah gene was done via hydrodynamic

293 injection of Cas9 nuclease, a sgRNA and a donor oligonucleotide that led to a significant step

294 towards gene therapy.112 VanLith et al. (2018) also showed hepatocyte-directed Fah gene

295 repair in HTI mice model using CRISPR/Cas9 against metabolic liver diseases.113

12
296 CRISPR/Cas9-based correction of Fah gene in HTI mice model showed weight stability

297 prevention of liver cirrhosis in mice.114

298 Hunter syndrome, a metabolic disorder, is caused by the mutational dysfunction of an

299 enzyme called iduronate-2-sulfatase (IDS), that leads to damages in lungs, heart and brain. At

300 the end of 2017, an in vivo genetic editing for the treatment of hunter syndrome via ZFNs was

301 reported.115 This is the first trial reporting the possibility of in vivo gene editing-mediated

302 treatment of genetic diseases. At the end of 2018, Sangamo Therapeutics in Richmond,

303 California, reported ZFNs-based in-body gene editing to in the people with hunter syndrome.

of
304 Although they obtained mixed results, no adverse side effects were observed in this

ro
305 therapy.116, 117
This initiative in gene-editing based therapy suggests the possible use of

306
-p
CRISPR/Cas9 for the treatment of metabolic disorders. Nevertheless, CRISPR/Cas9
re
307 technology against metabolic disorders needs efforts to enter into clinical trials.
lP

308
na

309 3.7. Cystic fibrosis

310 Cystic fibrosis (CF), an autosomal recessive monogenic condition, is caused by

ur

311 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene,118
Jo

312 causing damage to the lungs and digestive system. It has been noted that CRISPR/Cas9

313 approach may be a suitable method to correct the mutations in CFTR gene.119 Crane et al.

314 (2015) corrected the mutation of CFTR in iPSCs via CRISPR/Cas9 approach.120 In another

315 study, sheep models (CFTR-/- and CFTR+/-) were developed by CRISPR/Cas9-mediated

316 CFTR gene disruption to understand the CF pathogenesis.121 The mutated CFTR sheep model

317 is supposed to be a useful resource for the advanced development of new CF therapeutics. In

318 another study, CRISPR/Cas9 approach was used in the cultured stem cells of CF patients to

319 correct the CFTR gene.122 In primary adult stem cells, The CFTR gene alteration was done

13
320 through the homologous recombination.122 Therefore, CRISPR technology might be a

321 potential approach for the treatment of CF in future.

322

323 3.8. Blood-related disorders

324 Fanconi anaemia (FA), an autosomal recessive disorder, is caused by the mutations in

325 genes that are responsible for replication-dependent excision of interstrand DNA crosslink.123

326 CRISPR/Cas9-mediated homologous directed recombination (HDR) in FA gene implicate the

327 application of genome editing for correcting the defects in the DNA repair pathway.124

of
328 CRISPR/Cas9-mediated corrections of disruptive mutation in FA Complementation Group F

ro
329 (Fancf) gene,125 and correction of Fanconi anemia I (FANCI) gene126 were reported in iPSCs

330
-p
from primary fibroblasts. The correction of FA mutations is implicated with the therapeutic
re
331 approach of CRISPR/Cas9 against bone marrow failure.125 Richardson et al. (2018) found
lP

332 that human Cas9-induced single-strand template repair (SSTR) requires FA pathway for
na

333 DNA inter strand cross-link repair in cell.127

334 Sickle cell (SC) anemia, a blood-related disorders, is caused by a mutation in the β-
ur

335 globin gene that results in the formation of abnormal hemoglobin-S (HbS) protein.128 As
Jo

336 evidenced, CRISPR/Cas9 is safe and promising gene therapy approach for SC anemia.129-131

337 CRISPR/Cas9-based correction of Hbs gene in hematopoietic stem and progenitor cells

338 (HSPCs) from the SC disease patient blood confirmed normal functional reinstitution of

339 hemoglobin.132 In an in vitro study, CRISPR/Cas9 components showed more than 18% gene

340 modifications in CD34+ cells.133 They also reported correction of CD34+ HSPCs derived

341 from the bone marrow of SC anemia patients via CRISPR/Cas9 technology.133 Ye et al.

342 (2016) created hereditary persistence of fetal hemoglobin (HPFH) genotype in normal HSPCs

343 using CRISPR/Cas9 and indicated safe autologous transplantation for patients with sickle cell

344 disease and β-thalassemia.134

14
345 β-thalassemia, another widespread genetic blood-related disease, is caused by the

346 decreased synthesis of the β-globin chains of the hemoglobin tetramer which reduces the

347 production of hemoglobin.135, 136 Correction of β-thalassemia splice mutation (IVSII-1 G > A)

348 in iPSCs using Cas9 along with piggyBac transposon-modified donor vector strategy showed

349 repair of mutated gene with enhanced specificity and accuracy.137 This might be a critical

350 therapeutic approach toward the stem cell-based gene therapy against monogenic disorders in

351 future clinics.

352

of
353 3.9. Eye-related disorders

ro
354 Retinitis pigmentosa (RP), an inherited pigmentary retinal dystrophy, may causes loss

355
-p
of vision.138, 139 There are several mutation associated with this diseases, such as mutations in
re
356 RP1, rhodopsin (RHO) and retinitis pigmentosa GTPase regulator (RPGR) genes.140
lP

357 CRISPR/Cas9 gene editing technique can correct the Rho (S334) gene by sub retinal
na

358 injection of guide RNA/Cas9 plasmid, causing improvement of visual function through the

359 discontinuation of retinal degeneration in rats.141 Suzuki et al. (2016 & 2018) applied
ur

360 CRISPR/Cas9 using homology-independent targeted insertion (HITI), a method that allows
Jo

361 the targeted insertion of gene in non-dividing cells to improve the visual condition in rats.142,
143
362 This suggests the possibility of in vivo gene correction using CRISPR/Cas9 technology.

363 Several mutations at different genetic loci are reported in the cataract condition, i.e.

364 cloudiness of the crystalline lens.144 It has been noted that αA-crystallin gene mutations is

365 associated with an autosomal recessive cataract.145 To understand the role of the αA-

366 crystallin gene in congenital cataracts, Yuan et al. (2017) used CRISPR/Cas9 to develop

367 mutation in αA-crystallin gene in an animal model.146 Recently, it has been noted that

368 missense mutation of GJA8 is also associated with congenital cataract.147 Yuan et al. (2016)

369 have developed GJA8 knockout rabbit model using CRISPR/Cas9 system to study the

15
370 congenital cataracts in human.148 Currently, CRISPR/Cas9 mediated in vivo correction of

371 blindness condition is in clinical trial that is discussed later in this review.

372

373 3.10. Viral infection

374 3.10.1. Human immunodeficiency virus (HIV)

375 HIV is a pathogen that attacks human immune system and causes acquired

376 immunodeficiency syndrome (AIDS). Although AIDS is now manageable due to highly

377 active antiretroviral therapy (HAART), the life-long treatment of AIDS is of a great concern.

of
378 It has been noted that the expression of HIV-1 gene is induced by the long terminal repeats

ro
379 (LTRs), that are repeated identical sequences of DNA and assists in the insertion of retroviral

380
-p
DNA into the host chromosome. The genetic variation in the binding sites of LTRs may alter
re
381 LTR-driven viral transcription.149 It was observed that CRISPR/Cas9 can mutate LTRs in the
lP

382 DNA of HIV-1 provirus, leading to the breakdown of latent HIV-1 provirus.150 In a negative
na

383 feedback regulation of HIV-1, Cas9 gene was placed under the control of a minimal HIV-1

384 promoter to express Cas9 in HIV-1 contagious cells. This might also reduce the
ur

385 complications attributed by the unusual high expression of Cas9 in the cells.151 It has been
Jo

386 reported that CRISPR/Cas9-mediated gene editing can inhibit multiple steps of HIV-1

387 infection.152

388 Hultquist et al. (2019) used CRISPR/Cas9 technology for mechanistic examination of

389 HIV host factors in CD4+ T cells.153 Hartweger et al. (2019) edited B cells using

390 CRISPR/Cas9 in wild-type mouse to reduce the effect of HIV-1 infection.154 Some factors,

391 such as apolipoprotein B mRNA editing enzyme (APOBEC3G), and TRIM5α genes

392 expression, can cause host restriction against HIV infection. CRISPR/Cas9 technology

393 enhanced the expression of these host restriction factors against HIV infection,155, 156

16
394 suggesting the importance of CRISPR/Cas9-strategies as anti-HIV therapies. However, till

395 yet no curative therapy has been approved.

396

397

398 3.10.2. Hepatitis B virus (HBV)

399 HBV causes chronic hepatitis, which is a frequent infectious disease worldwide. It has

400 been observed that covalently closed circular DNAs (cccDNAs) of HBV reside in the

401 contaminated cells, suggesting cccDNA as a potential therapeutic target to treat HBV

of
402 infection.157-159 It was found that CRISPR/Cas9 nuclease can mediate interference of

ro
403 episomal cccDNA in reporter cell lines causing disruption in chromosomally integrated HBV

404
-p
sequences.160 Dong et al. (2015) demonstrated that CRISPR/Cas9 can reduces HBV cccDNA
re
405 by inhibiting viral replication.161 Ramanan et al. (2015) also showed that CRISPR/Cas9
lP

406 cleaves viral DNA and suppresses HBV.162 Wang et al. (2017) used RNA interference
na

407 (RNAi) technique using sgRNA-miRNA-gRNA cassette along with Cas9 to inhibit the

408 replication of HBV.163 Thus, it can be suggested that the disruption of the HBV genome
ur

409 through CRISPR/Cas9-technology might be a promising approach as an anti HBV therapy.

Jo

410

411 3.10.3. Hepatitis C virus (HCV)

412 HCV, a single-stranded RNA (ssRNA) virus, is the causative agent of hepatitis C, an

413 inflammatory condition of liver.164 It has been found that the Cas9 endonuclease enzyme

414 from the gram negative bacterium, Francisella novicida, known as FnCas9, can target

415 endogenous RNA.165 CRISPR/FnCas9 system has been used to inhibit HCV within

416 eukaryotic cells.166

417

418 3.10.4. Human papillomavirus (HPV)

17
419 HPV, a double-stranded DNA (dsDNA) virus, infects mucosal cells or skin. It causes

420 sexually transmitted disease and accounts for an estimated 11% of the global cancer

421 incidences in women.167,168 The RNA-guided endonuclease offers a therapeutic approach

422 against HPV.169 It was reported that the CRISPR/Cas9-mediated cervical cancer treatment

423 can be done by targeting HPVE6.170 CRISPR/Cas9 technology can also target the conserved

424 regions of HPV6/11 E7 genes, indicating the therapeutic potential of gene editing against

425 genital warts.171

426

of
427 3.10.5. Epstein−Barr virus (EBV)

ro
428 EBV, a dsDNA virus, spreads primarily through saliva and causes mononucleosis.172

429
-p
CRISPR/Cas9 technology has been used for genome editing of EBV in human cells by
re
430 modifying BART promoter gene encoding viral miRNAs.173 Ma et al. (2017) showed that 87
lP

431 LCL and 57 BL genes are significant for the survival and growth in lymphoblastoid cell lines
na

432 (LCLs) and BL cells.174 Ephrin receptor tyrosine kinase A2 (EphA2) facilitates the entry of

433 EBV in human cells. CRISPR/Cas9-mediated knockout experiments demonstrated that

ur

434 EphA2 extracellular domain can bind with the EBV- glycoprotein gHgL and provide the
Jo

435 entry to the cell.175 Thus, EphA2 might be a new potential target for therapeutic

436 development.

437

438 3.11. Translation of preclinical studies into clinical use

439 Although the preclinical studies on rodent models show evident efficiency of

440 CRISPR/Cas9-mediated genome editing, variable results may be obtained during clinical

441 translation of preclinical studies due to various reasons.176 Inadequate analysis of the

442 preclinical experimental data is one of the major reason for variable results.177 In addition,

443 presence of various backgrounds of most extensively used C57BL/6 and 129 strains of rodent

18
444 model might result in improper data analysis.178 Moreover, inbred mice cannot mimic the

445 diversity of humans.179 Another limitation of CRISPR/Cas9-mediate genome editing in

446 rodents is the precise knowledge of reproductive cycle of rodents, e.g. time of fertilization

447 and time to recover the fertilized eggs. Therefore, it is important to consider the limitations

448 and obtain in-depth knowledge of the animal model used in preclinical studies before

449 entering into clinical use.

450

of
451 4. Clinical trials

ro
452 China performed that first ex vivo clinical trial (clinicaltrials.gov; trial NCT02793856)

453
-p
employing gene editing using CRISPR/Cas9 technique in a patient with metastatic non-small-
re
454 cell lung cancer.180 They targeted PD-1 gene in T cells from peripheral blood of the patient
lP

455 using electroporation of sgRNA and Cas9 plasmid, and infused them back into the patient.
na

456 Very recently, they reported the presence of editied T-cells in the peripheral blood of all

457 patients who received infusion.181 They concluded that although this method is feasible and
ur

458 safe, more advanced gene editing technology is required to enhance the therapeutic efficacy.
Jo

459 Recently, Stadtmauer et al. reported the result of phase 1 in-human CRISPR/Cas9

460 technology-based clinical trial (clinicaltrials.gov; trial NCT03399448), performed in three

461 patients with refractory cancer in advanced stages.182 They removed TRAC and TRBC genes

462 that encodes the chains of endogenous TCR, and PDCD1 (encoding PD-1 loci) from the T

463 lymphocytes retrived from the patients to increase the anti-tumor immunity. They further

464 introduced transgene (NY-ESO-1) that can recognize tumor. These engineered T

465 lymphocytes were well tolerated by the patients upto 9 months after retroduction into the

466 patients.182 However chromosomal translocation was observed which was reduced after some

467 time.

19
468 Another clinical trial (clinicaltrials.gov; trial NCT03398967), proposed CAR-T

469 therapy for relapsed or refractory hematological malignancies due to CD19 negative tumor

470 cells. They performed integration of two CARs (i.e., CD19 and CD20 or CD22) in TRAC

471 locus of T-cells, that were able to recognize CD19 negative cells.183 In addition, the use of

472 gene-disrupted allogeneic universal CD19-specific CAR-T cell (UCART019) using lentivirus

473 (LV) delivery of CAR in patients with relapsed or refractory CD19+ leukemia and lymphoma

474 is also in clinical trial (clinicaltrials.gov; trial NCT03166878). In this study electroporation of

475 CRISPR RNA was used to disrupt endogenous TCR and B2M genes. This system could

of
476 minimize immunogenicity by avoiding graft-versus-host-disease (GVHD). However, the

ro
477 results are still not published. Most recently, the FDA approved clinical trial

478
-p
(clinicaltrials.gov; trial NCT04438083) of CTX130, an allogeneic CRISPR/Cas9-modified T
re
479 Cells, targeting CD70 against hematologic malignancies and renal cell carcinoma.
lP

480 In 2019, successful treatment of sickle-cell disease and β-thalassemia was reported by
na

481 Sangamo (clinicaltrials.gov; trial NCT03432364) via CRISPR/Cas9-mediated disruption of

ur

482 BCL11A gene in the stem cells that were isolated from patient’s peripheral blood with
Jo

483 hemoglobinopathies.184 BCL11A is a transcription regulator that suppress the expression of

484 β-globin gene. In another partial successful trial (clinicaltrials.gov; trial NCT03164135),

485 CCR5-deleted hematopoietic stem cells were transplanted in patient with HIV-1 and acute

486 lymphoblastic leukemia. Although the desired objective was not achieved, no major side

487 effects were observed.185 The study suggested the need for increasing the efficiency of CCR5

488 disruption in lymphocytes.185

489 In an in vivo clinical trial (clinicaltrials.gov; trial NCT03872479) registered in 2019,

490 CRISPR/Cas9 gene therapy-based drug AGN-151587 was given directly into the eye, via

491 subretinal injection, to cure a rare blindness condition called Leber’s congenital amaurosis 10

20
492 (LCA10) that is caused by mutations in the gene CEP290.186 This trial is the first approach to

493 deploy CRISPR/Cas9 gene editing therapy directly into the human body. At present, there are

494 approximately 19 registered interventional clinical trial on CRISPR/Cas9-mediated gene

495 editing technology (Table 2).

496

497 5. Challenges and future directions

498 Although both preclinical work and clinical trials focusing on curative therapies are

499 going on globally, the clinical translation of CRISPR/Cas9-mediated gene correction is

of
500 associated with unpredictability of the outcomes.187 Factors affecting the success rate of

ro
501 CRISPR/Cas9-mediated gene editing in humans includes off-target effects and cargo delivery

502
-p
methods. It has been observed that off-target effects are majorly guided by sgRNA, thus
re
503 rational designing of sgRNA is necessary to ensure the efficiency of CRISPR/Cas9 gene
lP

504 editing technology. It was observed that off-target were common in human cell culture with
na

505 persistent Cas9 expression.188, 189 while these effects were less common in in vivo models.190

506 It might be plausible that the occurrence of off-target effects in cell culture is due to the
ur

507 influence of various factors, such as cell type, expression level, transfection method, cell
Jo

508 culture maintenance, consecutive nuclease expression, guide sequence and repair events.191

509 Earlier, cuts at off-target sites that generates single stranded breaks (SSBs) in DNA

510 were consider as major obstacle and raised concern about the specificity of CRISPR/Cas9

511 technology.192 To reduce the possibility of SSB, paired CRISPR/Cas9 nickase that binds to

512 each forward and reverse DNA sequence on the flanking sides of target DNA was used to

513 generate DSB formation.193,194 Another approach suggested the use of inactive fusion protein

514 consisting of Cas9 and FokI endonuclease enzyme, which becomes functional only after

515 precise binding of sgRNA to both forward and reverse DNA sequences.195 Although these

516 approaches might reduce off-target cutting, there are certain limitations concerning

21
517 restrictions in the location of protospacer adjacent motifs (PAMs) near target DNA and

518 designing of sgRNAs for PAM alternatives.196 In addition, these approaches might also

519 increase the size of cargo that may generate constraint in the delivery system.

520 More importantly, repair events or a genomic rearrangement after sgRNAs-induced

521 DSBs is also a considerate safety concern in the CRISP/Cas9-based therapeutic interventions.

522 Although CRISPR/Cas9 technology can induce desired changes in the genomic sequences,

523 the poorly understood and less controlled DNA repair mechanism is associated with the

524 undesirable risk of biological dysfunctions. Deletion of few kilobases in the neighboring

of
525 CRISPR/Cas9 nickase activity, unexpected insertion (incorrect or partial) of donor DNA

ro
526 sequence to the site of integration and inversion are also unpredictable consequences of DNA

527
-p
repair mechanisms,197 that might result in unexpected mutations.198 The DSBs are repaired
re
528 via non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is a
lP

529 natural process to join spontaneous breaks in DNA and does not require any DNA template.
na

530 While, HDR requires either, donor DNA template or DNA that is synthesized by host’s

531 molecular recombination machinery. The NHEJ is an error prone mechanism that possibly
ur

532 leads to mutations. On the contrary, although HDR repairs DSBs more precisely, the
Jo

533 incidence of HDR is very low as compared to NHEJ.199 Various methods are suggested to

534 suppress NHEJ, i.e., chemical suppression of NHEJ,200 cell cycle synchronization,201

535 silencing of gene,202 and NHEJ-deficient cell lines.203 Therefore, enhancing the efficiency of

536 HDR and decreasing NHEJ is a critical challenge that needs to be fully explored before

537 entering into therapeutic regime to ensure the desired result of CRISPR/Cas-mediated gene

538 editing.

539 As the treatment of human diseases need to be tissue-specific, it is essential to

540 efficiently delivery the CRISPR/Cas9 cargo into target tissue. Therefore, additional concerns

541 should be given to the suitable delivery system that is based on the charge, size and the

22
542 content of the CRISPR/Cas9 cargo. CRISPR/Cas9 cargo might be of three types, i.e., plasmid

543 DNA encoding sgRNA and Cas9, combination of sgRNA and Cas9 mRNA and combination

544 of sgRNA and Cas9 protein. Various physical, viral and non-viral systems have been used as

545 a vector for the delivery of CRISPR/Cas9.

546 AAVs and LVs are the most commonly used viral vectors for gene editing.204 The

547 advantages of AAVs includes less immunogenic property, serotype specificity, good safety

548 profile and ability to transduce both dividing and non-dividing cells.205 However, mild

549 toxicity is reported at high doses in animal models.206 In addition, LVs also has the advantage

of
550 of high transducing efficiency and non-immunogenicity. Moreover, it has the ability to be

ro
551 pseudotyped, allowing alterations in the LVs cellular tropism. Another important aspect to

552
-p
consider while using viral delivery vectors is their packing limitation. However, it was
re
553 observed that the carrying capacity of AAVs can be extended up to 35 kb after modifications.
lP

554 It might be possible that CRISPR/Cas9 gene editing complex can activate the host immune
na

555 response system207, indicating the need for modifications that can help to avoid immune

556 response in the host.

ur

557 In addition to viral delivery systems, the use of non-viral systems or nanoparticles
Jo

558 (NPs) for the delivery of CRISPR/Cas9 is also suggested.207 Lipofectamine is the

559 commercially available lipid NPs for sgRNA and Cas9 delivery in various systems.208, 209

560 The cationic property of lipid NPs might allow better packaging of anionic Cas9/sgRNA

561 complex in the NPs. As observed, lipid NPs have low delivery efficiency due to its reduced

562 uptake by the cells, low translocation in the nucleus and the possible entrapment of NPs in

563 the endosomes. However, the use of optimal lipids for the synthesis of lipid NPs, surface

564 modification by cell targeting agent, avoidance of immune system and additional of

565 endosomal escape system can enhance the delivery efficiency of liposomes.210

23
566 Various other non-liposomal delivering techniques are also suggested for the delivery
211
567 of Cas9 and sgRNA, i.e., FuGENE-6 reagent based on electrostatic interactions , calcium

568 phosphate transfection212, cell-penetrating peptides213, DNA nanoclew214, inorganic NPs215,

569 and polyethenimine and poly(L-lysine) (PLL) polymers. PLL is used to develop a therapeutic

570 delivery system, called MENDs, consisting of plasmid DNA and lipid shell.216 Further

571 modifications in MENDs might allow the targeting of delivery cargo to mitochondria and

572 nucleus of the cells.217 Although MENDs showed high transfection rate, in vivo validation of

573 this delivery system is still requisite. Therefore, most of the delivery methods for

of
574 CRISPR/Cas9 system have both advantages and disadvantages. This generates a need for

ro
575 extensive work on the development of suitable delivery system with high specificity, high

576
-p
transfection rate, high capacity and low immunogenicity. Moreover, long-term safety and low
re
577 toxicity associated with any delivery system also need consideration before developing
lP

578 CRISPR/Cas9-based gene editing therapy against human diseases.24

na

579

580 6. Conclusions
ur

581 The forthcoming clinical application of CRISPR/Cas9 is promising for clinical world.
Jo

582 With the progression of genome-editing techniques, the genome editing research related to

583 therapy of human diseases using CRISPR/Cas9 is developing quickly. Just few years before,

584 this technology was started and presently a huge number of scientists working on this

585 technology. Among them, most of the researches are using this genome editing technology

586 for the therapy of human diseases.

587 However, CRISPR/Cas9 is still a budding technology that is applied to patients with

588 severe conditions at life threating stage. Most of the clinical trials are currently in phase I/II.

589 Till now, the clinical trials are focusing on the safety and efficacy of genome editing in

590 humans, to improve molecular processes involved in genome editing. The therapeutic gene

24
591 editing technology is now expanding to ethically controversial alteration in the genome of

592 human-early stage embryos to protect from HIV infection.218 Such attempts can also suggest

593 scientific community to establish regulations on gene-editing. However, the journey of

594 CRISPR/Cas9 is highly interesting and it offers a significant hope for the researchers for the

595 treatment of human deadly diseases.

596

597 AUTHOR CONTRIBUTIONS

598 Conceptualization, C.C. and S.S.L.; Writing – Original Draft, C.C., G.S.; A.R.S; Writing –

of
599 Review and Editing, A.R.S. and M.B.; Supervision and Funding, S.S.L. and A.R.S.

ro
600

601 CONFLICTS OF INTEREST

-p
re
602 Authors declare no conflict of interests.
lP

603
na

604 ACKNOWLEDGMENTS

605 This study was supported by Hallym University Research Fund and by Basic Science
ur

606 Research Program through the National Research Foundation of Korea (NRF) funded by the
Jo

607 Ministry of Education (NRF -2017R1A2B4012944 & NRF-2020R1C1C1008694).

608

609 References
610 1. Young, CS, Pyle, AD, and Spencer, MJ (2019). CRISPR for neuromuscular disorders:

611 gene editing and beyond. Physiology 34: 341-353.

612 2. Chakraborty, C, Teoh, SL, and Das, S (2017). The smart programmable CRISPR

613 technology: A next generation genome editing tool for investigators. Current drug

614 targets 18: 1653-1663.

25
615 3. Hsu, PD, Lander, ES, and Zhang, F (2014). Development and applications of

616 CRISPR-Cas9 for genome engineering. Cell 157: 1262-1278.

617 4. Peng, R, Lin, G, and Li, J (2016). Potential pitfalls of CRISPR/Cas9‐mediated

618 genome editing. The FEBS journal 283: 1218-1231.

619 5. Bortesi, L, and Fischer, R (2015). The CRISPR/Cas9 system for plant genome editing

620 and beyond. Biotechnology advances 33: 41-52.

621 6. Ryan, OW, Poddar, S, and Cate, JH (2016). Crispr–cas9 genome engineering in

622 Saccharomyces cerevisiae cells. Cold Spring Harbor Protocols 2016: pdb.

of
623 prot086827.

ro
624 7. Friedland, AE, Tzur, YB, Esvelt, KM, Colaiácovo, MP, Church, GM, and Calarco, JA

625
-p
(2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature
re
626 methods 10: 741-743.
lP

627 8. Gratz, SJ, Rubinstein, CD, Harrison, MM, Wildonger, J, and O'Connor‐Giles, KM
na

628 (2015). CRISPR‐Cas9 genome editing in Drosophila. Current protocols in molecular

629 biology 111: 31.32. 31-31.32. 20.

ur

630 9. Liu, J, Zhou, Y, Qi, X, Chen, J, Chen, W, Qiu, G, et al. (2017). CRISPR/Cas9 in
Jo

631 zebrafish: an efficient combination for human genetic diseases modeling. Human

632 genetics 136: 1-12.

633 10. Platt, RJ, Chen, S, Zhou, Y, Yim, MJ, Swiech, L, Kempton, HR, et al. (2014).

634 CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159: 440-

635 455.

636 11. Zhu, S, Zhou, Y, and Wei, W (2017). Genome-wide CRISPR/Cas9 screening for

637 high-throughput functional genomics in human cells. Innate Antiviral Immunity.

638 Springer. pp 175-181.

26
639 12. Pennisi, E (2013). The CRISPR craze. American Association for the Advancement of

640 Science.

641 13. Chylinski, K, Makarova, KS, Charpentier, E, and Koonin, EV (2014). Classification

642 and evolution of type II CRISPR-Cas systems. Nucleic Acids Res 42: 6091-6105.

643 14. Brouns, SJ, Jore, MM, Lundgren, M, Westra, ER, Slijkhuis, RJ, Snijders, AP, et al.

644 (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:

645 960-964.

646 15. Porteus, MH (2019a). A New Class of Medicines through DNA Editing. N Engl J

of
647 Med 380: 947-959.

ro
648 16. Porteus, MH (2019b). A new class of medicines through DNA editing. New England

649 Journal of Medicine 382: 577-583.

-p
re
650 17. Cong, L, Ran, FA, Cox, D, Lin, S, Barretto, R, Habib, N, et al. (2013). Multiplex
lP

651 genome engineering using CRISPR/Cas systems. Science 339: 819-823.

na

652 18. Mali, P, Yang, L, Esvelt, KM, Aach, J, Guell, M, DiCarlo, JE, et al. (2013). RNA-

653 guided human genome engineering via Cas9. Science 339: 823-826.
ur

654 19. Baylis, F, and McLeod, M (2017). First-in-human phase 1 CRISPR gene editing
Jo

655 cancer trials: are we ready? Current gene therapy 17: 309-319.

656 20. Cyranoski, D (2016). Chinese scientists to pioneer first human CRISPR trial. Nature

657 News 535: 476.

658 21. Schaefer, KA, Wu, W-H, Colgan, DF, Tsang, SH, Bassuk, AG, and Mahajan, VB

659 (2017). Unexpected mutations after CRISPR–Cas9 editing in vivo. Nature methods

660 14: 547.

661 22. Check, E (2002). A tragic setback. Nature 420: 116-118.

662 23. Kaiser, J (2003). Gene therapy. Seeking the cause of induced leukemias in X-SCID

663 trial. Science 299: 495.

27
664 24. Lino, CA, Harper, JC, Carney, JP, and Timlin, JA (2018). Delivering CRISPR: a

665 review of the challenges and approaches. Drug Deliv 25: 1234-1257.

666 25. Gaspar, HB, Cooray, S, Gilmour, KC, Parsley, KL, Adams, S, Howe, SJ, et al.

667 (2011). Long-term persistence of a polyclonal T cell repertoire after gene therapy for

668 X-linked severe combined immunodeficiency. Sci Transl Med 3: 97ra79.

669 26. Hacein-Bey-Abina, S, Hauer, J, Lim, A, Picard, C, Wang, GP, Berry, CC, et al.

670 (2010). Efficacy of gene therapy for X-linked severe combined immunodeficiency. N

671 Engl J Med 363: 355-364.

of
672 27. Marshall, E (1999). Gene therapy death prompts review of adenovirus vector. Science

ro
673 286: 2244-2245.

674 28.
-p
Bibikova, M, Beumer, K, Trautman, JK, and Carroll, D (2003). Enhancing gene
re
675 targeting with designed zinc finger nucleases. Science 300: 764.
lP

676 29. Christian, M, Cermak, T, Doyle, EL, Schmidt, C, Zhang, F, Hummel, A, et al. (2010).
na

677 Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:

678 757-761.
ur

679 30. Jansen, R, Embden, JDv, Gaastra, W, and Schouls, LM (2002). Identification of genes
Jo

680 that are associated with DNA repeats in prokaryotes. Molecular microbiology 43:

681 1565-1575.

682 31. Wolfe, SA, Nekludova, L, and Pabo, CO (2000). DNA recognition by Cys2His2 zinc

683 finger proteins. Annu Rev Biophys Biomol Struct 29: 183-212.

684 32. Doyon, Y, Vo, TD, Mendel, MC, Greenberg, SG, Wang, J, Xia, DF, et al. (2011).

685 Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric

686 architectures. Nat Methods 8: 74-79.

28
687 33. Ramirez, CL, Foley, JE, Wright, DA, Muller-Lerch, F, Rahman, SH, Cornu, TI, et al.

688 (2008). Unexpected failure rates for modular assembly of engineered zinc fingers. Nat

689 Methods 5: 374-375.

690 34. Muenzer, J, Prada, CE, Burton, B, Lau, HA, Ficicioglu, C, Foo, CWP, et al. (2019).

691 CHAMPIONS: A phase 1/2 clinical trial with dose escalation of SB-913 ZFN-

692 mediated in vivo human genome editing for treatment of MPS II (Hunter syndrome).

693 Molecular Genetics and Metabolism 126: S104.

694 35. Cannon, P, and June, C (2011). Chemokine receptor 5 knockout strategies. Curr Opin

of
695 HIV AIDS 6: 74-79.

ro
696 36. Joung, JK, and Sander, JD (2013). TALENs: a widely applicable technology for

697
-p
targeted genome editing. Nature reviews Molecular cell biology 14: 49-55.
re
698 37. Bhattacharya, D, Marfo, CA, Li, D, Lane, M, and Khokha, MK (2015).
lP

699 CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease
na

700 genes in Xenopus. Developmental biology 408: 196-204.

701 38. Yang, H, Wu, J-J, Tang, T, Liu, K-D, and Dai, C (2017). CRISPR/Cas9-mediated
ur

702 genome editing efficiently creates specific mutations at multiple loci using one
Jo

703 sgRNA in Brassica napus. Scientific reports 7: 1-13.

704 39. Komor, AC, Kim, YB, Packer, MS, Zuris, JA, and Liu, DR (2016). Programmable

705 editing of a target base in genomic DNA without double-stranded DNA cleavage.

706 Nature 533: 420-424.

707 40. Jemal, A, Bray, F, Center, MM, Ferlay, J, Ward, E, and Forman, D (2011). Global

708 cancer statistics. CA: a cancer journal for clinicians 61: 69-90.

709 41. Hanahan, D, and Weinberg, RA (2000). The hallmarks of cancer. Cell 100: 57-70.

29
710 42. Alfarouk, KO, Stock, C-M, Taylor, S, Walsh, M, Muddathir, AK, Verduzco, D, et al.

711 (2015). Resistance to cancer chemotherapy: failure in drug response from ADME to

712 P-gp. Cancer cell international 15: 71.

713 43. Hanahan, D, and Weinberg, RA (2011). Hallmarks of cancer: the next generation.

714 Cell 144: 646-674.

715 44. Sánchez-Rivera, FJ, and Jacks, T (2015). Applications of the CRISPR–Cas9 system in

716 cancer biology. Nature reviews cancer 15: 387-395.

717 45. Liu, Y, Zeng, Y, Liu, L, Zhuang, C, Fu, X, Huang, W, et al. (2014). Synthesizing

of
718 AND gate genetic circuits based on CRISPR-Cas9 for identification of bladder cancer

ro
719 cells. Nature communications 5: 1-7.

720 46.
-p
Murati, A, Brecqueville, M, Devillier, R, Mozziconacci, M-J, Gelsi-Boyer, V, and
re
721 Birnbaum, D (2012). Myeloid malignancies: mutations, models and management.
lP

722 BMC cancer 12: 304.

na

723 47. Valletta, S, Dolatshad, H, Bartenstein, M, Yip, BH, Bello, E, Gordon, S, et al. (2015).

724 ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells
ur

725 and increases survival in mouse xenografts. Oncotarget 6: 44061.

Jo

726 48. Aubrey, BJ, Kelly, GL, Kueh, AJ, Brennan, MS, O’Connor, L, Milla, L, et al. (2015).

727 An inducible lentiviral guide RNA platform enables the identification of tumor-

728 essential genes and tumor-promoting mutations in vivo. Cell reports 10: 1422-1432.

729 49. Craig, R (2002). MCL1 provides a window on the role of the BCL2 family in cell

730 proliferation, differentiation and tumorigenesis. Leukemia 16: 444-454.

731 50. Niu, Y, Xu, J, and Sun, T (2019). Cyclin-dependent kinases 4/6 inhibitors in breast

732 cancer: Current status, resistance, and combination strategies. Journal of Cancer 10:

733 5504.

30
734 51. Zhou, Y, Han, C, Li, D, Yu, Z, Li, F, Li, F, et al. (2015). Cyclin-dependent kinase 11

735 p110 (CDK11 p110) is crucial for human breast cancer cell proliferation and growth.

736 Scientific reports 5: 1-13.

737 52. Feng, Y, Sassi, S, Shen, JK, Yang, X, Gao, Y, Osaka, E, et al. (2015). Targeting

738 Cdk11 in osteosarcoma cells using the CRISPR‐cas9 system. Journal of orthopaedic

739 research 33: 199-207.

740 53. Wang, Y, Zhang, T, Kwiatkowski, N, Abraham, BJ, Lee, TI, Xie, S, et al. (2015).

741 CDK7-dependent transcriptional addiction in triple-negative breast cancer. Cell 163:

of
742 174-186.

ro
743 54. Liu, T, Li, Z, Zhang, Q, Bernstein, KDA, Lozano-Calderon, S, Choy, E, et al. (2016).

744
-p
Targeting ABCB1 (MDR1) in multi-drug resistant osteosarcoma cells using the
re
745 CRISPR-Cas9 system to reverse drug resistance. Oncotarget 7: 83502.
lP

746 55. Tang, H, and Shrager, JB (2016). CRISPR/Cas‐mediated genome editing to treat
na

747 EGFR‐mutant lung cancer: a personalized molecular surgical therapy. EMBO

748 molecular medicine 8: 83-85.

ur

749 56. Feng, W, Li, H-c, Xu, K, Chen, Y-f, Pan, L-y, Mei, Y, et al. (2016). SHCBP1 is over-
Jo

750 expressed in breast cancer and is important in the proliferation and apoptosis of the

751 human malignant breast cancer cell line. Gene 587: 91-97.

752 57. Dhanoa, BS, Cogliati, T, Satish, AG, Bruford, EA, and Friedman, JS (2013). Update

753 on the Kelch-like (KLHL) gene family. Human genomics 7: 1-7.

754 58. Lian, Y-F, Yuan, J, Cui, Q, Feng, Q-S, Xu, M, Bei, J-X, et al. (2016). Upregulation of

755 KLHDC4 predicts a poor prognosis in human nasopharyngeal carcinoma. PLoS One

756 11: e0152820.

757 59. Birling, MC, Herault, Y, and Pavlovic, G (2017). Modeling human disease in rodents

758 by CRISPR/Cas9 genome editing. Mamm Genome 28: 291-301.

31
759 60. Birling, MC, Schaeffer, L, Andre, P, Lindner, L, Marechal, D, Ayadi, A, et al. (2017).

760 Efficient and rapid generation of large genomic variants in rats and mice using

761 CRISMERE. Sci Rep 7: 43331.

762 61. Chen, S, Sanjana, NE, Zheng, K, Shalem, O, Lee, K, Shi, X, et al. (2015). Genome-

763 wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160:

764 1246-1260.

765 62. Matano, M, Date, S, Shimokawa, M, Takano, A, Fujii, M, Ohta, Y, et al. (2015).

766 Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human

of
767 intestinal organoids. Nature medicine 21: 256-262.

ro
768 63. Zuckermann, M, Hovestadt, V, Knobbe-Thomsen, CB, Zapatka, M, Northcott, PA,

769
-p
Schramm, K, et al. (2015). Somatic CRISPR/Cas9-mediated tumour suppressor
re
770 disruption enables versatile brain tumour modelling. Nature communications 6: 1-9.
lP

771 64. Heckl, D, Kowalczyk, MS, Yudovich, D, Belizaire, R, Puram, RV, McConkey, ME,
na

772 et al. (2014). Generation of mouse models of myeloid malignancy with combinatorial

773 genetic lesions using CRISPR-Cas9 genome editing. Nature biotechnology 32: 941-
ur

774 946.
Jo

775 65. Maude, SL, Laetsch, TW, Buechner, J, Rives, S, Boyer, M, Bittencourt, H, et al.

776 (2018). Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic

777 Leukemia. N Engl J Med 378: 439-448.

778 66. Neelapu, SS, Locke, FL, Bartlett, NL, Lekakis, LJ, Miklos, DB, Jacobson, CA, et al.

779 (2017). Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell

780 Lymphoma. N Engl J Med 377: 2531-2544.

781 67. Schuster, SJ, Bishop, MR, Tam, CS, Waller, EK, Borchmann, P, McGuirk, JP, et al.

782 (2019). Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell

783 Lymphoma. N Engl J Med 380: 45-56.

32
784 68. Vormittag, P, Gunn, R, Ghorashian, S, and Veraitch, FS (2018). A guide to

785 manufacturing CAR T cell therapies. Curr Opin Biotechnol 53: 164-181.

786 69. Seimetz, D, Heller, K, and Richter, J (2019). Approval of first CAR-Ts: have we

787 solved all hurdles for ATMPs? Cell Medicine 11: 2155179018822781.

788 70. Fried, LP, and Bush, TL (1988). Morbidity as a focus of preventive health care in the

789 elderly. Epidemiol Rev 10: 48-64.

790 71. Park, JH, Riviere, I, Gonen, M, Wang, X, Senechal, B, Curran, KJ, et al. (2018).

791 Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N

of
792 Engl J Med 378: 449-459.

ro
793 72. Rapoport, AP, Stadtmauer, EA, Binder-Scholl, GK, Goloubeva, O, Vogl, DT, Lacey,

794
-p
SF, et al. (2015). NY-ESO-1-specific TCR-engineered T cells mediate sustained
re
795 antigen-specific antitumor effects in myeloma. Nat Med 21: 914-921.
lP

796 73. Goodman, MA, Moradi Manesh, D, Malik, P, and Rothenberg, ME (2017).
na

797 CRISPR/Cas9 in allergic and immunologic diseases. Taylor & Francis.

798 74. Wu, Q, Case, SR, Minor, MN, Jiang, D, Martin, RJ, Bowler, RP, et al. (2013). A
ur

799 novel function of MUC18: amplification of lung inflammation during bacterial

Jo

800 infection. The American journal of pathology 182: 819-827.

801 75. Chu, HW, Rios, C, Huang, C, Wesolowska-Andersen, A, Burchard, EG, O’Connor,

802 BP, et al. (2015). CRISPR–Cas9-mediated gene knockout in primary human airway

803 epithelial cells reveals a proinflammatory role for MUC18. Gene therapy 22: 822-

804 829.

805 76. Su, S, Hu, B, Shao, J, Shen, B, Du, J, Du, Y, et al. (2016). CRISPR-Cas9 mediated

806 efficient PD-1 disruption on human primary T cells from cancer patients. Scientific

807 reports 6: 20070.

33
808 77. Arason, G, Jorgensen, G, and Ludviksson, B (2010). Primary immunodeficiency and

809 autoimmunity: lessons from human diseases. Scandinavian journal of immunology 71:

810 317-328.

811 78. Kuo, CY, Hoban, MD, Joglekar, AV, and Kohn, DB (2015). Site specific Gene

812 correction of defects in CD40 ligand using the Crispr/Cas9 genome editing platform.

813 Journal of Allergy and Clinical Immunology 135: AB17.

814 79. Cheong, T-C, Compagno, M, and Chiarle, R (2016). Editing of mouse and human

815 immunoglobulin genes by CRISPR-Cas9 system. Nature communications 7: 1-10.

of
816 80. Malaviya, R, Laskin, DL, and Malaviya, R (2010). Janus kinase-3 dependent

ro
817 inflammatory responses in allergic asthma. International immunopharmacology 10:

818 829-836.
-p
re
819 81. Chang, C-W, Lai, Y-S, Westin, E, Khodadadi-Jamayran, A, Pawlik, KM, Lamb Jr,
lP

820 LS, et al. (2015). Modeling human severe combined immunodeficiency and
na

821 correction by CRISPR/Cas9-enhanced gene targeting. Cell reports 12: 1668-1677.

822 82. Min, Y-L, Li, H, Rodriguez-Caycedo, C, Mireault, AA, Huang, J, Shelton, JM, et al.
ur

823 (2019). CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion

Jo

824 mutations in mice and human cells. Science advances 5: eaav4324.

825 83. Rooney, JE, Welser, JV, Dechert, MA, Flintoff-Dye, NL, Kaufman, SJ, and Burkin,

826 DJ (2006). Severe muscular dystrophy in mice that lack dystrophin and α7 integrin.

827 Journal of cell science 119: 2185-2195.

828 84. Guo, C, Willem, M, Werner, A, Raivich, G, Emerson, M, Neyses, L, et al. (2006).

829 Absence of α7 integrin in dystrophin-deficient mice causes a myopathy similar to

830 Duchenne muscular dystrophy. Human molecular genetics 15: 989-998.

34
831 85. Tabebordbar, M, Zhu, K, Cheng, JK, Chew, WL, Widrick, JJ, Yan, WX, et al. (2016).

832 In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351:

833 407-411.

834 86. Nelson, CE, Hakim, CH, Ousterout, DG, Thakore, PI, Moreb, EA, Rivera, RMC, et

835 al. (2016). In vivo genome editing improves muscle function in a mouse model of

836 Duchenne muscular dystrophy. Science 351: 403-407.

837 87. Long, C, Amoasii, L, Mireault, AA, McAnally, JR, Li, H, Sanchez-Ortiz, E, et al.

838 (2016). Postnatal genome editing partially restores dystrophin expression in a mouse

of
839 model of muscular dystrophy. Science 351: 400-403.

ro
840 88. Long, C, McAnally, JR, Shelton, JM, Mireault, AA, Bassel-Duby, R, and Olson, EN

841
-p
(2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing
re
842 of germline DNA. Science 345: 1184-1188.
lP

843 89. Ousterout, DG, Kabadi, AM, Thakore, PI, Majoros, WH, Reddy, TE, and Gersbach,
na

844 CA (2015). Multiplex CRISPR/Cas9-based genome editing for correction of

845 dystrophin mutations that cause Duchenne muscular dystrophy. Nature

ur

846 communications 6: 1-13.

Jo

847 90. Van Agtmaal, EL, André, LM, Willemse, M, Cumming, SA, Van Kessel, ID, Van

848 Den Broek, WJ, et al. (2017). CRISPR/Cas9-induced (CTG⋅ CAG) n repeat instability

849 in the myotonic dystrophy type 1 locus: implications for therapeutic genome editing.

850 Molecular Therapy 25: 24-43.

851 91. Lee, K, Conboy, M, Park, HM, Jiang, F, Kim, HJ, Dewitt, MA, et al. (2017).

852 Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces

853 homology-directed DNA repair. Nature biomedical engineering 1: 889-901.

35
854 92. Zhang, Y, Li, H, Min, YL, Sanchez-Ortiz, E, Huang, J, Mireault, AA, et al. (2020).

855 Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a

856 self-complementary AAV delivery system. Science advances 6: eaay6812.

857 93. Bergeron, N, Phan, BAP, Ding, Y, Fong, A, and Krauss, RM (2015). Proprotein

858 convertase subtilisin/kexin type 9 inhibition: a new therapeutic mechanism for

859 reducing cardiovascular disease risk. Circulation 132: 1648-1666.

860 94. Seidah, NG (2013). Proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors in the

861 treatment of hypercholesterolemia and other pathologies. Current pharmaceutical

of
862 design 19: 3161-3172.

ro
863 95. Jiang, C, Mei, M, Li, B, Zhu, X, Zu, W, Tian, Y, et al. (2017). A non-viral

864
-p
CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in
re
865 vivo. Cell research 27: 440-443.
lP

866 96. Jarrett, KE, Lee, C, De Giorgi, M, Hurley, A, Gillard, BK, Doerfler, AM, et al.
na

867 (2018). Somatic editing of Ldlr with adeno-associated viral-CRISPR is an efficient

868 tool for atherosclerosis research. Arteriosclerosis, thrombosis, and vascular biology
ur

869 38: 1997-2006.

Jo

870 97. Tessadori, F, Roessler, HI, Savelberg, SM, Chocron, S, Kamel, SM, Duran, KJ, et al.

871 (2018). Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human

872 genetic cardiovascular disorders. Disease models & mechanisms 11.

873 98. Saudou, F, and Humbert, S (2016). The biology of huntingtin. Neuron 89: 910-926.

874 99. Yang, S, Chang, R, Yang, H, Zhao, T, Hong, Y, Kong, HE, et al. (2017).

875 CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of

876 Huntington’s disease. The Journal of clinical investigation 127: 2719-2724.

36
877 100. Shin, JW, Kim, K-H, Chao, MJ, Atwal, RS, Gillis, T, MacDonald, ME, et al. (2016).

878 Permanent inactivation of Huntington's disease mutation by personalized allele-

879 specific CRISPR/Cas9. Human molecular genetics 25: 4566-4576.

880 101. Monteys, AM, Ebanks, SA, Keiser, MS, and Davidson, BL (2017). CRISPR/Cas9

881 editing of the mutant huntingtin allele in vitro and in vivo. Molecular Therapy 25: 12-

882 23.

883 102. Levy-Lahad, E, Wijsman, EM, Nemens, E, Anderson, L, Goddard, K, Weber, JL, et

884 al. (1995). A familial Alzheimer's disease locus on chromosome 1. Science 269: 970-

of
885 973.

ro
886 103. Pires, C, Schmid, B, Petræus, C, Poon, A, Nimsanor, N, Nielsen, TT, et al. (2016).

887
-p
Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease
re
888 patient iPSC line carrying a A79V mutation in PSEN1. Stem cell research 17: 285-
lP

889 288.
na

890 104. Poon, A, Schmid, B, Pires, C, Nielsen, TT, Hjermind, LE, Nielsen, JE, et al. (2016).

891 Generation of a gene-corrected isogenic control hiPSC line derived from a familial
ur

892 Alzheimer's disease patient carrying a L150P mutation in presenilin 1. Stem cell
Jo

893 research 17: 466-469.

894 105. György, B, Lööv, C, Zaborowski, MP, Takeda, S, Kleinstiver, BP, Commins, C, et al.

895 (2018). CRISPR/Cas9 mediated disruption of the Swedish APP allele as a therapeutic

896 approach for early-onset Alzheimer’s disease. Molecular Therapy-Nucleic Acids 11:

897 429-440.

898 106. Rohn, TT, Kim, N, Isho, NF, and Mack, JM (2018). The Potential of CRISPR/Cas9

899 Gene Editing as a Treatment Strategy for Alzheimer's Disease. Journal of Alzheimer's

900 disease & Parkinsonism 8.

37
901 107. Villiger, L, Grisch-Chan, HM, Lindsay, H, Ringnalda, F, Pogliano, CB, Allegri, G, et

902 al. (2018). Treatment of a metabolic liver disease by in vivo genome base editing in

903 adult mice. Nature medicine 24: 1519-1525.

904 108. Singh, K, Evens, H, Nair, N, Rincón, MY, Sarcar, S, Samara-Kuko, E, et al. (2018).

905 Efficient in vivo liver-directed gene editing using CRISPR/Cas9. Molecular Therapy

906 26: 1241-1254.

907 109. Yang, Y, Wang, L, Bell, P, McMenamin, D, He, Z, White, J, et al. (2016). A dual

908 AAV system enables the Cas9-mediated correction of a metabolic liver disease in

of
909 newborn mice. Nature biotechnology 34: 334-338.

ro
910 110. Morrow, G, and Tanguay, RM (2017). Biochemical and clinical aspects of hereditary

911
-p
tyrosinemia type 1. Hereditary Tyrosinemia. Springer. pp 9-21.
re
912 111. Nasrallah, F, Hammami, MB, Rhouma, B, Fradj, SH, Azzouz, H, Omar, S, et al.
lP

913 (2015). Clinical and Biochemical Profile of Tyrosinemia Type 1 in Tunisia. Clinical
na

914 laboratory 61: 487-492.

915 112. Yin, H, Xue, W, Chen, S, Bogorad, RL, Benedetti, E, Grompe, M, et al. (2014).
ur

916 porteu editing with Cas9 in adult mice corrects a disease mutation and phenotype.
Jo

917 Nature biotechnology 32: 551-553.

918 113. VanLith, C, Guthman, R, Nicolas, CT, Allen, K, Du, Z, Joo, DJ, et al. (2018).

919 Curative ex vivo hepatocyte-directed gene editing in a mouse model of hereditary

920 tyrosinemia type 1. Human gene therapy 29: 1315-1326.

921 114. Shao, Y, Wang, L, Guo, N, Wang, S, Yang, L, Li, Y, et al. (2018). Cas9-nickase–

922 mediated genome editing corrects hereditary tyrosinemia in rats. Journal of Biological

923 Chemistry 293: 6883-6892.

38
924 115. Li, H, Yang, Y, Hong, W, Huang, M, Wu, M, and Zhao, X (2020). Applications of

925 genome editing technology in the targeted therapy of human diseases: mechanisms,

926 advances and prospects. Signal Transduct Target Ther 5: 1.

927 116. Ledford, H (2018). First test of in-body gene editing shows promise. Nature 10.

928 117. Kaiser, J (2018). New gene-editing treatment might help treat a rare disorder, hints

929 first human test. Science 10.

930 118. Skov, M, Hansen, CR, and Pressler, T (2019). Cystic fibrosis–an example of

931 personalized and precision medicine. Apmis 127: 352-360.

of
932 119. Marangi, M, and Pistritto, G (2018). Innovative therapeutic strategies for cystic

ro
933 fibrosis: moving forward to CRISPR technique. Frontiers in pharmacology 9: 396.

934 120.
-p
Crane, AM, Kramer, P, Bui, JH, Chung, WJ, Li, XS, Gonzalez-Garay, ML, et al.
re
935 (2015). Targeted correction and restored function of the CFTR gene in cystic fibrosis
lP

936 induced pluripotent stem cells. Stem cell reports 4: 569-577.

na

937 121. Fan, Z, Perisse, IV, Cotton, CU, Regouski, M, Meng, Q, Domb, C, et al. (2018). A

938 sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR
ur

939 gene. JCI insight 3.

Jo

940 122. Schwank, G, Koo, B-K, Sasselli, V, Dekkers, JF, Heo, I, Demircan, T, et al. (2013).

941 Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic

942 fibrosis patients. Cell stem cell 13: 653-658.

943 123. Moldovan, G-L, and D'Andrea, AD (2009). How the fanconi anemia pathway guards

944 the genome. Annual review of genetics 43: 223-249.

945 124. Osborn, MJ, Gabriel, R, Webber, BR, DeFeo, AP, McElroy, AN, Jarjour, J, et al.

946 (2015). Fanconi anemia gene editing by the CRISPR/Cas9 system. Human gene

947 therapy 26: 114-126.

39
948 125. Skvarova Kramarzova, K, Osborn, MJ, Webber, BR, DeFeo, AP, McElroy, AN, Kim,

949 CJ, et al. (2017). CRISPR/Cas9-mediated correction of the FANCD1 gene in primary

950 patient cells. International journal of molecular sciences 18: 1269.

951 126. Osborn, MJ, Lonetree, C-l, Webber, BR, Patel, D, Dunmire, S, DeFeo, AP, et al.

952 (2016). CRISPR/Cas9 targeted gene editing and cellular engineering in Fanconi

953 anemia. Stem cells and development 25: 1591-1603.

954 127. Richardson, CD, Kazane, KR, Feng, SJ, Zelin, E, Bray, NL, Schäfer, AJ, et al. (2018).

955 CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia

of
956 pathway. Nature genetics 50: 1132-1139.

ro
957 128. Williams, TN, and Thein, SL (2018). Sickle cell anemia and its phenotypes. Annual

958
-p
review of genomics and human genetics 19: 113-147.
re
959 129. Demirci, S, Leonard, A, Haro-Mora, JJ, Uchida, N, and Tisdale, JF (2019).
lP

960 CRISPR/Cas9 for sickle cell disease: applications, future possibilities, and challenges.
na

961 Cell Biology and Translational Medicine, Volume 5. Springer. pp 37-52.

962 130. Tasan, I, Jain, S, and Zhao, H (2016). Use of genome-editing tools to treat sickle cell
ur

963 disease. Human genetics 135: 1011-1028.

Jo

964 131. Sato, M, Saitoh, I, and Inada, E (2016). Efficient CRISPR/Cas9-based gene correction

965 in induced pluripotent stem cells established from fibroblasts of patients with sickle

966 cell disease. Stem cell investigation 3.

967 132. Wen, J, Tao, W, Hao, S, and Zu, Y (2017). Cellular function reinstitution of offspring

968 red blood cells cloned from the sickle cell disease patient blood post CRISPR genome

969 editing. Journal of hematology & oncology 10: 1-11.

970 133. Hoban, MD, Lumaquin, D, Kuo, CY, Romero, Z, Long, J, Ho, M, et al. (2016).

971 CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells.

972 Molecular Therapy 24: 1561-1569.

40
973 134. Ye, L, Wang, J, Tan, Y, Beyer, AI, Xie, F, Muench, MO, et al. (2016). Genome

974 editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for

975 treating sickle cell disease and β-thalassemia. Proceedings of the National Academy

976 of Sciences 113: 10661-10665.

977 135. Xie, F, Ye, L, Chang, JC, Beyer, AI, Wang, J, Muench, MO, et al. (2014). Seamless

978 gene correction of β-thalassemia mutations in patient-specific iPSCs using

979 CRISPR/Cas9 and piggyBac. Genome research 24: 1526-1533.

980 136. Cao, A, and Galanello, R (2010). Beta-thalassemia. Genetics in medicine 12: 61-76.

of
981 137. Alateeq, S, Ovchinnikov, D, Tracey, T, Whitworth, D, Al-Rubaish, A, Al-Ali, A, et

ro
982 al. (2018). Identification of on-target mutagenesis during correction of a beta-

983
-p
thalassemia splice mutation in iPS cells with optimised CRISPR/Cas9-double nickase
re
984 reveals potential safety concerns. APL bioengineering 2: 046103.
lP

985 138. Vezinaw, CM, Fishman, GA, and McAnany, JJ (2020). Visual Impairment in Retinitis
na

986 Pigmentosa. Retina.

987 139. Shintani, K, Shechtman, DL, and Gurwood, AS (2009). Review and update: current
ur

988 treatment trends for patients with retinitis pigmentosa. Optometry-Journal of the
Jo

989 American Optometric Association 80: 384-401.

990 140. Wang, D, Chan, W, Tam, P, Baum, L, Lam, D, Chong, K, et al. (2005). Gene

991 mutations in retinitis pigmentosa and their clinical implications. Clinica chimica acta

992 351: 5-16.

993 141. Bakondi, B, Lv, W, Lu, B, Jones, MK, Tsai, Y, Kim, KJ, et al. (2016). In vivo

994 CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of

995 autosomal dominant retinitis pigmentosa. Molecular Therapy 24: 556-563.

996 142. Suzuki, K, and Belmonte, JCI (2018). In vivo genome editing via the HITI method as

997 a tool for gene therapy. Journal of Human Genetics 63: 157-164.

41
 998 143. Suzuki, K, Tsunekawa, Y, Hernandez-Benitez, R, Wu, J, Zhu, J, Kim, EJ, et al.

999 (2016). In vivo genome editing via CRISPR/Cas9 mediated homology-independent

1000 targeted integration. Nature 540: 144-149.

1001 144. Shiels, A, and Hejtmancik, JF (2017). Mutations and mechanisms in congenital and

1002 age-related cataracts. Exp Eye Res 156: 95-102.

1003 145. Shiels, A, and Hejtmancik, JF (2007). Genetic origins of cataract. Arch Ophthalmol

1004 125: 165-173.

1005 146. Yuan, L, Yao, H, Xu, Y, Chen, M, Deng, J, Song, Y, et al. (2017). CRISPR/Cas9-

of
1006 Mediated Mutation of alphaA-Crystallin Gene Induces Congenital Cataracts in

ro
1007 Rabbits. Invest Ophthalmol Vis Sci 58: BIO34-BIO41.

1008 147.
-p
Hadrami, M, Bonnet, C, Veten, F, Zeitz, C, Condroyer, C, Wang, P, et al. (2019). A
re
1009 novel missense mutation of GJA8 causes congenital cataract in a large Mauritanian
lP

1010 family. Eur J Ophthalmol 29: 621-628.

na

1011 148. Yuan, L, Sui, T, Chen, M, Deng, J, Huang, Y, Zeng, J, et al. (2016). CRISPR/Cas9-

1012 mediated GJA8 knockout in rabbits recapitulates human congenital cataracts. Sci Rep
ur

1013 6: 22024.
Jo

1014 149. Shah, S, Alexaki, A, Pirrone, V, Dahiya, S, Nonnemacher, MR, and Wigdahl, B

1015 (2014). Functional properties of the HIV-1 long terminal repeat containing single-

1016 nucleotide polymorphisms in Sp site III and CCAAT/enhancer binding protein site I.

1017 Virol J 11: 92.

1018 150. Ebina, H, Misawa, N, Kanemura, Y, and Koyanagi, Y (2013). Harnessing the

1019 CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci Rep 3: 2510.

1020 151. Kaminski, R, Chen, Y, Salkind, J, Bella, R, Young, WB, Ferrante, P, et al. (2016).

1021 Negative Feedback Regulation of HIV-1 by Gene Editing Strategy. Sci Rep 6: 31527.

42
1022 152. Yin, L, Hu, S, Mei, S, Sun, H, Xu, F, Li, J, et al. (2018). CRISPR/Cas9 Inhibits

1023 Multiple Steps of HIV-1 Infection. Hum Gene Ther 29: 1264-1276.

1024 153. Hultquist, JF, Hiatt, J, Schumann, K, McGregor, MJ, Roth, TL, Haas, P, et al. (2019).

1025 CRISPR-Cas9 genome engineering of primary CD4(+) T cells for the interrogation of

1026 HIV-host factor interactions. Nat Protoc 14: 1-27.

1027 154. Hartweger, H, McGuire, AT, Horning, M, Taylor, JJ, Dosenovic, P, Yost, D, et al.

1028 (2019). HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells. J

1029 Exp Med 216: 1301-1310.

of
1030 155. Bogerd, HP, Kornepati, AV, Marshall, JB, Kennedy, EM, and Cullen, BR (2015).

ro
1031 Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived

1032
-p
transcriptional activators. Proc Natl Acad Sci U S A 112: E7249-7256.
re
1033 156. Dufour, C, Claudel, A, Joubarne, N, Merindol, N, Maisonnet, T, Masroori, N, et al.
lP

1034 (2018). Editing of the human TRIM5 gene to introduce mutations with the potential to
na

1035 inhibit HIV-1. PLoS One 13: e0191709.

1036 157. Zhu, A, Liao, X, Li, S, Zhao, H, Chen, L, Xu, M, et al. (2019). HBV cccDNA and Its
ur

1037 Potential as a Therapeutic Target. J Clin Transl Hepatol 7: 258-262.

Jo

1038 158. Bloom, K, Maepa, MB, Ely, A, and Arbuthnot, P (2018). Gene Therapy for Chronic

1039 HBV-Can We Eliminate cccDNA? Genes (Basel) 9.

1040 159. Lee, C (2019). CRISPR/Cas9-Based Antiviral Strategy: Current Status and the

1041 Potential Challenge. Molecules 24.

1042 160. Karimova, M, Beschorner, N, Dammermann, W, Chemnitz, J, Indenbirken, D,

1043 Bockmann, JH, et al. (2015). CRISPR/Cas9 nickase-mediated disruption of hepatitis

1044 B virus open reading frame S and X. Sci Rep 5: 13734.

43
1045 161. Dong, C, Qu, L, Wang, H, Wei, L, Dong, Y, and Xiong, S (2015). Targeting hepatitis

1046 B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication.

1047 Antiviral Res 118: 110-117.

1048 162. Ramanan, V, Shlomai, A, Cox, DB, Schwartz, RE, Michailidis, E, Bhatta, A, et al.

1049 (2015). CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus.

1050 Sci Rep 5: 10833.

1051 163. Wang, J, Chen, R, Zhang, R, Ding, S, Zhang, T, Yuan, Q, et al. (2017). The gRNA-

1052 miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach

of
1053 Strongly Inhibits Hepatitis B Virus Replication. Theranostics 7: 3090-3105.

ro
1054 164. Li, H, Huang, MH, Jiang, JD, and Peng, ZG (2018). Hepatitis C: From inflammatory

1055
-p
pathogenesis to anti-inflammatory/hepatoprotective therapy. World J Gastroenterol
re
1056 24: 5297-5311.
lP

1057 165. Sampson, TR, Saroj, SD, Llewellyn, AC, Tzeng, YL, and Weiss, DS (2013). A
na

1058 CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature

1059 497: 254-257.

ur

1060 166. Price, AA, Sampson, TR, Ratner, HK, Grakoui, A, and Weiss, DS (2015). Cas9-
Jo

1061 mediated targeting of viral RNA in eukaryotic cells. Proc Natl Acad Sci U S A 112:

1062 6164-6169.

1063 167. McMurray, HR, Nguyen, D, Westbrook, TF, and McAnce, DJ (2001). Biology of

1064 human papillomaviruses. Int J Exp Pathol 82: 15-33.

1065 168. Sanclemente, G, and Gill, DK (2002). Human papillomavirus molecular biology and

1066 pathogenesis. J Eur Acad Dermatol Venereol 16: 231-240.

1067 169. Kennedy, EM, Kornepati, AV, Goldstein, M, Bogerd, HP, Poling, BC, Whisnant,

1068 AW, et al. (2014). Inactivation of the human papillomavirus E6 or E7 gene in cervical

44
1069 carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. J Virol

1070 88: 11965-11972.

1071 170. Yoshiba, T, Saga, Y, Urabe, M, Uchibori, R, Matsubara, S, Fujiwara, H, et al. (2019).

1072 CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus

1073 E6. Oncol Lett 17: 2197-2206.

1074 171. Liu, YC, Cai, ZM, and Zhang, XJ (2016). Reprogrammed CRISPR-Cas9 targeting the

1075 conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis

1076 in E7-transformed keratinocytes. Asian J Androl 18: 475-479.

of
1077 172. Hurt, C, and Tammaro, D (2007). Diagnostic evaluation of mononucleosis-like

ro
1078 illnesses. Am J Med 120: 911 e911-918.

1079 173.
-p
Yuen, KS, Chan, CP, Wong, NM, Ho, CH, Ho, TH, Lei, T, et al. (2015).
re
1080 CRISPR/Cas9-mediayoungted genome editing of Epstein-Barr virus in human cells. J
lP

1081 Gen Virol 96: 626-636.

na

1082 174. Ma, Y, Walsh, MJ, Bernhardt, K, Ashbaugh, CW, Trudeau, SJ, Ashbaugh, IY, et al.

1083 (2017). CRISPR/Cas9 Screens Reveal Epstein-Barr Virus-Transformed B Cell Host

ur

1084 Dependency Factors. Cell Host Microbe 21: 580-591 e587.

Jo

1085 175. Chen, J, Sathiyamoorthy, K, Zhang, X, Schaller, S, Perez White, BE, Jardetzky, TS,

1086 et al. (2018). Ephrin receptor A2 is a functional entry receptor for Epstein-Barr virus.

1087 Nat Microbiol 3: 172-180.

1088 176. Justice, MJ, and Dhillon, P (2016). Using the mouse to model human disease:

1089 increasing validity and reproducibility. Dis Model Mech 9: 101-103.

1090 177. Kafkafi, N, Golani, I, Jaljuli, I, Morgan, H, Sarig, T, Wurbel, H, et al. (2017).

1091 Addressing reproducibility in single-laboratory phenotyping experiments. Nat

1092 Methods 14: 462-464.

45
1093 178. Simpson, EM, Linder, CC, Sargent, EE, Davisson, MT, Mobraaten, LE, and Sharp, JJ

1094 (1997). Genetic variation among 129 substrains and its importance for targeted

1095 mutagenesis in mice. Nat Genet 16: 19-27.

1096 179. Bilovocky, NA, Romito-DiGiacomo, RR, Murcia, CL, Maricich, SM, and Herrup, K

1097 (2003). Factors in the genetic background suppress the engrailed-1 cerebellar

1098 phenotype. J Neurosci 23: 5105-5112.

1099 180. Cyranoski, D (2016). CRISPR gene-editing tested in a person for the first time.

1100 Nature 539: 479.

of
1101 181. Lu, Y, Xue, J, Deng, T, Zhou, X, Yu, K, Deng, L, et al. (2020). Safety and feasibility

ro
1102 of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat

1103 Med 26: 732-740.

-p
re
1104 182. Stadtmauer, EA, Fraietta, JA, Davis, MM, Cohen, AD, Weber, KL, Lancaster, E, et
lP

1105 al. (2020). CRISPR-engineered T cells in patients with refractory cancer. Science 367:
na

1106 eaba7365.

1107 183. Fousek, K, Watanabe, J, Joseph, SK, George, A, An, X, Byrd, TT, et al. (2020). CAR
ur

1108 T-cells that target acute B-lineage leukemia irrespective of CD19 expression.
Jo

1109 Leukemia.

1110 184. The, LH (2019). CRISPR-Cas9 gene editing for patients with haemoglobinopathies.

1111 The Lancet Haematology 6: e438.

1112 185. Xu, L, Wang, J, Liu, Y, Xie, L, Su, B, Mou, D, et al. (2019). CRISPR-edited stem

1113 cells in a patient with HIV and acute lymphocytic leukemia. New England Journal of

1114 Medicine 381: 1240-1247.

1115 186. Ledford, H (2020). CRISPR treatment inserted directly into the body for first time.

1116 Nature 579: 185.

46
1117 187. Teboul, L, Herault, Y, Wells, S, Qasim, W, and Pavlovic, G (2020). Variability in

1118 genome editing outcomes: challenges for research reproducibility and clinical safety.

1119 Molecular Therapy.

1120 188. Fu, Y, Foden, JA, Khayter, C, Maeder, ML, Reyon, D, Joung, JK, et al. (2013). High-

1121 frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.

1122 Nat Biotechnol 31: 822-826.

1123 189. Wang, X, Wang, Y, Wu, X, Wang, J, Qiu, Z, Chang, T, et al. (2015). Unbiased

1124 detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-

of
1125 defective lentiviral vectors. Nat Biotechnol 33: 175-178.

ro
1126 190. Iyer, V, Boroviak, K, Thomas, M, Doe, B, Riva, L, Ryder, E, et al. (2018). No

1127
-p
unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-
re
1128 edited mice. PLoS Genet 14: e1007503.
lP

1129 191. Teboul, L, Herault, Y, Wells, S, Qasim, W, and Pavlovic, G (2020). Variability in
na

1130 Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical

1131 Safety. Mol Ther 28: 1422-1431.

ur

1132 192. Tsai, SQ, Zheng, Z, Nguyen, NT, Liebers, M, Topkar, VV, Thapar, V, et al. (2015).
Jo

1133 GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas

1134 nucleases. Nat Biotechnol 33: 187-197.

1135 193. Ran, FA, Hsu, PD, Lin, CY, Gootenberg, JS, Konermann, S, Trevino, AE, et al.

1136 (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing

1137 specificity. Cell 154: 1380-1389.

1138 194. Cho, SW, Kim, S, Kim, Y, Kweon, J, Kim, HS, Bae, S, et al. (2014). Analysis of off-

1139 target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.

1140 Genome Res 24: 132-141.

47
1141 195. Tsai, SQ, Wyvekens, N, Khayter, C, Foden, JA, Thapar, V, Reyon, D, et al. (2014).

1142 Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat

1143 Biotechnol 32: 569-576.

1144 196. Han, HA, Pang, JKS, and Soh, BS (2020). Mitigating off-target effects in

1145 CRISPR/Cas9-mediated in vivo gene editing. J Mol Med (Berl) 98: 615-632.

1146 197. Kraft, K, Geuer, S, Will, AJ, Chan, WL, Paliou, C, Borschiwer, M, et al. (2015).

1147 Deletions, Inversions, Duplications: Engineering of Structural Variants using

1148 CRISPR/Cas in Mice. Cell Rep 10: 833-839.

of
1149 198. Simeonov, DR, Brandt, AJ, Chan, AY, Cortez, JT, Li, Z, Woo, JM, et al. (2019). A

ro
1150 large CRISPR-induced bystander mutation causes immune dysregulation. Commun

1151 Biol 2: 70.

-p
re
1152 199. Maruyama, T, Dougan, SK, Truttmann, MC, Bilate, AM, Ingram, JR, and Ploegh, HL
lP

1153 (2015). Increasing the efficiency of precise genome editing with CRISPR-Cas9 by
na

1154 inhibition of nonhomologous end joining. Nat Biotechnol 33: 538-542.

1155 200. Vartak, SV, and Raghavan, SC (2015). Inhibition of nonhomologous end joining to
ur

1156 increase the specificity of CRISPR/Cas9 genome editing. FEBS J 282: 4289-4294.
Jo

1157 201. Lin, SR, Yang, HC, Kuo, YT, Liu, CJ, Yang, TY, Sung, KC, et al. (2014). The

1158 CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In

1159 Vivo. Mol Ther Nucleic Acids 3: e186.

1160 202. Chu, VT, Weber, T, Wefers, B, Wurst, W, Sander, S, Rajewsky, K, et al. (2015).

1161 Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced

1162 precise gene editing in mammalian cells. Nat Biotechnol 33: 543-548.

1163 203. Seelos, HJ (1992). A new paradigm of medical informatics. Methods Inf Med 31: 79-

1164 81.

48
1165 204. Xu, CL, Ruan, MZC, Mahajan, VB, and Tsang, SH (2019). Viral Delivery Systems

1166 for CRISPR. Viruses 11.

1167 205. Yi, L, and Li, J (2016). CRISPR-Cas9 therapeutics in cancer: promising strategies and

1168 present challenges. Biochim Biophys Acta 1866: 197-207.

1169 206. Lau, CH, and Suh, Y (2017). In vivo genome editing in animals using AAV-CRISPR

1170 system: applications to translational research of human disease. F1000Research 6:

1171 2153.

1172 207. Kim, S, Koo, T, Jee, HG, Cho, HY, Lee, G, Lim, DG, et al. (2018). CRISPR RNAs

of
1173 trigger innate immune responses in human cells. Genome Res.

ro
1174 208. Yu, X, Liang, X, Xie, H, Kumar, S, Ravinder, N, Potter, J, et al. (2016). Improved

1175
-p
delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX.
re
1176 Biotechnology letters 38: 919-929.
lP

1177 209. Berardo, C, Siciliano, V, Di Pasqua, LG, Richelmi, P, Vairetti, M, and Ferrigno, A
na

1178 (2019). Comparison between Lipofectamine RNAiMAX and GenMute transfection

1179 agents in two cellular models of human hepatoma. European Journal of

ur

1180 Histochemistry: EJH 63.

Jo

1181 210. Wang, M, Zuris, JA, Meng, F, Rees, H, Sun, S, Deng, P, et al. (2016). Efficient

1182 delivery of genome-editing proteins using bioreducible lipid nanoparticles.

1183 Proceedings of the National Academy of Sciences 113: 2868-2873.

1184 211. Kennedy, EM, Kornepati, AV, Goldstein, M, Bogerd, HP, Poling, BC, Whisnant,

1185 AW, et al. (2014). Inactivation of the human papillomavirus E6 or E7 gene in cervical

1186 carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. Journal

1187 of virology 88: 11965-11972.

49
1188 212. Jin, W, Lin, D, Nguyen, AH, Abdelrasoul, GN, Chen, J, Mar, A, et al. (2018).

1189 Transfection of difficult-to-transfect rat primary cortical neurons with magnetic

1190 nanoparticles. Journal of biomedical nanotechnology 14: 1654-1664.

1191 213. Suresh, B, Ramakrishna, S, and Kim, H (2017). Cell-penetrating peptide-mediated

1192 delivery of Cas9 protein and guide RNA for genome editing. Eukaryotic

1193 Transcriptional and Post-Transcriptional Gene Expression Regulation. Springer. pp

1194 81-94.

1195 214. Sun, W, Ji, W, Hall, JM, Hu, Q, Wang, C, Beisel, CL, et al. (2015). Self‐assembled

of
1196 DNA nanoclews for the efficient delivery of CRISPR–Cas9 for genome editing.

ro
1197 Angewandte Chemie 127: 12197-12201.

1198 215.
-p
Wang, P, Zhang, L, Zheng, W, Cong, L, Guo, Z, Xie, Y, et al. (2018). Thermo‐
re
1199 triggered release of CRISPR‐Cas9 system by lipid‐encapsulated gold nanoparticles
lP

1200 for tumor therapy. Angewandte Chemie International Edition 57: 1491-1496.
na

1201 216. Kogure, K, Moriguchi, R, Sasaki, K, Ueno, M, Futaki, S, and Harashima, H (2004).

1202 Development of a non-viral multifunctional envelope-type nano device by a novel

ur

1203 lipid film hydration method. Journal of controlled release 98: 317-323.
Jo

1204 217. Nakamura, T, Akita, H, Yamada, Y, Hatakeyama, H, and Harashima, H (2012). A

1205 multifunctional envelope-type nanodevice for use in nanomedicine: concept and

1206 applications. Accounts of chemical research 45: 1113-1121.

1207 218. Rose, BI, and Brown, S (2019). Genetically Modified Babies and a First Application

1208 of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9).

1209 Obstetrics & Gynecology 134: 157-162.

1210

1211

1212

50
1213 Figure legend:

1214 Figure 1. Timeline of the breakthrough and progression of CRISPR/Cas9 systems.

1215 Figure 2. CRISPR/Cas9 system dealing for treatment of multiple human diseases.

1216

1217 Table legends:

1218 Table 1. CRISPR/Cas9 research and its application for the treatment of the different human

1219 diseases.

1220 Table 2. Currently registered interventional clinical trials with CRISPR/Cas9-based gene

of
1221 editing (clinicaltrials.gov; search date: 2020/07/05).

ro
1222

1223
-p
1224
re
lP
na
ur
Jo

51
1225 Table 1: CRISPR/Cas9 research and its application for the treatment of the different human

1226 diseases

Disease type Disease related Remark References

Gene/Protein
47
ASXL1 (additional sex- CRISPR/Cas9 was used to decrease

comb like-1) leukemia cell growth in mouse

xenografts.
48
Mcl-1 (myeloid cell CRISPR/Cas9 was used to delete
Cancer

of
leukemia 1) MCL-1 in human BL cells and

ro
induce the apoptosis in the BL cells.

Cyclin dependent kinase -pCRISPR/Cas9 was used to silence 52

re
11(CDK11) the Cyclin dependent kinase
lP

11(CDK11) in osteosarcoma.
56
SHC SH2-binding protein 1 SHCBP1 inhibits the proliferation of
na

(SHCBP1) breast cancer through CRISPR/Cas9

ur

58
Kelch domain containing 4 CRISPR/Cas9 was used to knockout
Jo

(KLHDC4) KLHDC4 gene in a nasopharyngeal

carcinoma cell line

55
Epidermal Growth Factor CRISPR/Cas9 was used for possible

Receptor (EGFR) correction of acquired drug resistant

mutations in EGFR
75
Allergy Melanoma cell adhesion CRISPR/Cas9 technology uses to

molecule knockout cell surface glycoprotein

(MCAM/ MUC18) MUC18 in human primary nasal

airway epithelial cells

52
 80
JAK3 (Janus Kinase 3) CRISPR/Cas9 was used to re-

establish the development of normal

T-cell in JAK3-deficient cells

88
Duchenne muscular Dystrophin CRISPR/Cas9 was used to fix DMD

dystrophy gene mutation for the Duchenne

muscular dystrophy disorder

94
Cardiovascular Proprotein convertase CRISPR/Cas9 was used to correct

diseases subtilisin/kexin type 9 the PCSK9 gene in atherosclerosis

of
(PCSK9) mouse model

ro
99
Huntington disease Huntingtin (HTT)gene -p
CRISPR/Cas9 was used to suppress
re
the mHTT gene selectively in

mouse model
lP

103,106
Alzheimer’s Presenilin 1 (PSEN1) and CRISPR/Cas9 was used to correct
na

Disease Presenilin 2 (PSEN2) gene ancestral mutations in AD related to

ur

the PSEN gene

Jo

Metabolic liver Pahenu2 CRISPR/Cas9 was used to correct 107

disease Pahenu2 gene in metabolic liver

disease
124
Fanconi anaemia 17 Fanconi anaemia (FA) CRISPR/Cas9 was used to correct

the Fanconianemia
114
Hereditary Fumarylacetoacetase (Fah) CRISPR/Cas9 was used to correct

tyrosinemia the Fah mutation in mouse models

132
Sickle cell anemia β-globin gene CRISPR/Cas9 was used to edit treat

sickle cell disease patient blood

53
 135
Β-Thalassemia Hemoglobin Subunit Beta CRISPR/Cas9 was used to corrected

(HBB) gene HBB gene mutation in human

iPSCs from β-thalassemia patients

122
Cystic fibrosis Cystic fibrosis CRISPR/Cas9 was used to correct

transmembrane the CFTR gene in cultured stem

conductance regulator cells of Cystic fibrosis patients

(CFTR) gene

141
Retinitis RP1,RHO, and RPGR gene CRISPR/Cas9 was used to interrupt

of
pigmentosa the Rho(S334) mutation

ro
146
Cataract αA-crystallin gene -p
CRISPR/Cas9 was used tyo study
re
the relationship of αA-crystallin

mutations and human congenital

lP

cataracts
na

150
Human Long terminal repeats CRISPR/Cas9 was used as tool to
ur

immunodeficiency (LTRs) in HIV mutate LTRs of HIV-1 DNA

Jo

virus (HIV)

161
Hepatitis B virus covalently closed circular CRISPR/Cas9 was used to target

(HBV) DNAs (cccDNAs) in HBV cccDNA of HBV

170
Human papilloma HPVE6 gene CRISPR/Cas9 was used target to

virus (HPV) HPVE6 for cancer treatment

175
Epstein−Barr virus Ephrin receptor tyrosine Using EphA2 extracellular domain,

(EBV) kinase A2 (EphA2) therapeutic strategy was developed

1227
1228

54
1229 Table 2. Currently registered interventional clinical trials with CRISPR/Cas9-based gene

1230 editing (clinicaltrials.gov; search date: 2020/07/05).

Sl. NCT Target gene Disease Intervention Phase Study Country

no number and effect start date

1. NCT0442666 Cytokine- Gastro- Tumor I/II May 15, USA

9 induced SH2 Intestinal Infiltrating 2020
(CISH) protein (GI) Cancer. Lymphocytes
inhibition (TIL)

of
inhibited

ro
immune
-p checkpoint
CISH
re
2. NCT0417838 Target Severe Detection of - August 1, China
lP

2 adjustment of Sepsis alveolar 2019

antibiotics lavage fluid
na

changes the
choice of early
ur

antibiotics in
Jo

patients with
pneumonia
3. NCT0403756 Disruption of Refractory B CD19-CAR I August China
6 HPK1 cell modified T 2019
malignancie cells with
s CAR
delivered by
lentivirus and
Cas9 knockout
of HPK1
4. NCT0316413 CCR5 knockout HIV Modified - May 30, China
5 CD34+ 2017
hematopoietic

55
 stem cells

5. NCT0354581 Programmed Mesothelin CAR T cells I June 1, China

5 cell death positive to mesothelin 2018
protein 1 (PD- solid tumors with added
1) and TCR PD-1 and TCR
knockout knockout
6. NCT0305791 E6 and E7 HPV-related Plasmid in a I January 15, China
2 oncogene of malignancy gel containing 2018

of
HPV16 and a polymer to
HPV18 deletion facilitate

ro
delivery
7. NCT0334254 Stem cell- Gastrointesti -pDuodenal - April 18, China
re
7 derived human nal Infection biopsies, 2018
intestinal followed by
lP

enteroids differentiation
into mini-guts
na

8. NCT0365567 Disruption of β- Ex vivo I/II September U.K.,

8 the erythroid
ur

thalassemia modified 14, 2018 German

enhancer to hematopoietic y,
Jo

BCL11A gene stem cells Canada,

Italy
9. NCT0372832 Correction of β- Ex vivo I January Not
2 the thalassemia modified 2019 specified
hemoglobulin hematopoietic
subunit β stem cells
globulin gene
10. NCT0424465 CTX120 B-cell Multiple Biological I January 22, USA,
6 maturation Myeloma safety and 2020 Spain,
antigen efficacy of Australi
(BCMA)- CTX120 in a
directed T-cell multiple

56
 immunotherapy myeloma

11. NCT0443808 CTX130 CD70- Renal Cell Safety and I June 16, Australi
3 directed T-cell Carcinoma efficacy of 2020 a
immunotherapy CTX130 in
comprised of relapsed or
allogeneic T refractory
cells renal cell
carcinoma.

of
12. NCT0374796 Programmed Mesothelin CAR T cells I November China
5 cell death positive to mesothelin 2018

ro
protein 1 (PD- solid tumors with PD-1
1) knockout -p
knockout
re
13. NCT0339896 Cas9-mediated B Cell CAR T cells I/II January 2, China
7 creation of Leukemia to CD19 and 2018
lP

CD19 and CD20 or

CD20 or CD19 CD19 and
na

and CD22 CD22

CAR-T cells
ur

14. NCT0403543 Creation of a Refractory CD19-directed I/II July 22, U.S.A.,

Jo

4 CD19-directed B-cell T-cell 2019 Australi

T cell malignancie immunotherap a
s y
15. NCT0374528 Disruption of Sickle cell Ex vivo I/II November U.S.A.
7 the erythroid anemia modified 27, 2018
enhancer to hematopoietic
BCL11A gene stem cells
16. NCT0316687 βTCRα, TCRβ, B-cell CD19-CAR I/II June 2017 China
8 β-2 microglobin leukemia modified T
(B2M) cells with
knockout CAR
delivered by

57
 lentivirus and
Cas9 knockout
B2M and TCR
to create
universal T
cells
17. NCT0304474 Programmed EBV- Modified T I/II April 7, China
3 cell death positive, cells selected 2017
protein 1 (PD- advanced for those
1) knockout stage targeting EBV

of
malignancie positive cells

ro
s
18. NCT0441776 PD-1 knockout Hepatocellul TACE I June 20, China
4 engineered T ar
-p combined 2019
re
cells Carcinoma treatment to
block the
lP

blood supply
na

of the tumor
19. NCT0387247 Removal of Leber ZFN-mediated I September U.S.A.
ur

9 alternative congenital removal of 26, 2019

splice site in amaurosis intronic
Jo

CEP290 10 alternative
splice site in
retinal cells
1231
1232

58
eTOC Synopsis :

CRISPR/Cas9 genome editing tool showed promising advancements in clinical trials. However,

the results of CRISPR/Cas9-based genome editing is unpredictable and raises safety concerns.

Sharma et al., review the preclinical and clinical evidence for CRISPR/Cas9-mediated genome

editing and discuss their potential challenges and future strategies to minimize the limitations of

CRISPR/Cas9.

of
ro
-p
re
lP
na
ur
Jo
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of