DNA targeting specificity of RNA-guided Cas9 nucleases

Hsu, Patrick; Scott, David; Weinstein, Joshua A.; Ran, F. Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J.; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J.; Marraffini, Luciano A.; Bao, Gang; Zhang, Feng

doi:10.1038/nbt.2647

Cited by 4,155 publications

(4,464 citation statements)

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“…Cas9-Venus C-terminal fusion protein (Cas9-CV) showed similar sgRNA-dependent in vivo cleavage activity on its genomic target as untagged wild type protein, while N-terminal Venus-Cas9 (NV-Cas9) fusion showed no activity (Supplementary information, Figure S1A). In addition, Cas9-CV also showed similar cleavage activity on a previously identified emx1 off-target (emx1-OT4) [9,11] but did not cleave the control locus (Supplementary information, Figure S1B-S1D). Mutations of both HNH nuclease and RuvC catalytic domains (DM-Cas9-CV) abolished the cleavage activity (Supplementary information, Figure S1A, lane5).…”

mentioning

confidence: 61%

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

et al. 2014

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mentioning

confidence: 61%

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

et al. 2014

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“…Earlier works have studied the specificity of the gRNA design. Although Cong et al reported highly specific gRNA targeting and abolished gRNA function, caused by mismatches at the last 11 bases on the 20‐mer‐targeting region,2 subsequent work indicated that any mismatches on the 20‐mer‐targeting region of gRNA might cause Cas9 off‐targeting 29. With the optimized micelle, no significant editing was observed in the off‐targeting sites identified by both Cas‐OFFinder and BLAST (off/on ratio ≈0; Figure 3F,G).…”

Section: Discussionmentioning

confidence: 99%

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Lao

Gao

et al. 2018

Advanced Science

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CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

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“…Apart from the overall targeting and HDR efficacy and mosaicism, one safety concern regarding clinical application of gene correction in human embryos is that CRISPR-Cas9 can induce undesirable off-target mutations at genome regions that are highly homologous to the targeted sequence [22][23][24] . Therefore, we conducted a comprehensive, whole-genome In the S-phase-injected group, each mosaic embryo (green) contained blastomeres with different genotypes.…”

Section: Off-target Consequences In Repaired Human Embryosmentioning

confidence: 99%

Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

819

540

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More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms. Genes in which dominant mutations manifest as late-onset adult disorders include BRCA1 and BRCA2, which are associated with a high risk of breast and ovarian cancers 1 , and MYBPC3, mutation of which causes hypertrophic cardiomyopathy (HCM) 2 . Because of their delayed manifestation, these mutations escape natural selection and are often transmitted to the next generation. Consequently, the frequency of some of these founder mutations in particular human populations is very high. For example, the MYBPC3 mutation is found at frequencies ranging from 2% to 8% 3 in major Indian populations, and the estimated frequency of both BRCA1 and BRCA2 mutations among Ashkenazi Jews exceeds 2% 4 .HCM is a myocardial disease characterized by left ventricular hypertrophy, myofibrillar disarray and myocardial stiffness; it has an estimated prevalence of 1:500 in adults 5 and manifests clinically with heart failure. HCM is the commonest cause of sudden death in otherwise healthy young athletes. HCM, while not a uniformly fatal condition, has a tremendous impact on the lives of individuals, including physiological (heart failure and arrhythmias), psychological (limited activity and fear of sudden death), and genealogical concerns. MYBPC3 mutations account for approximately 40% of all genetic defects causing HCM and are also responsible for a large fraction of other inherited cardiomyopathies, including dilated cardiomyopathy and left ventricular non-compaction 6 . MYBPC3 encodes the thick filament-associated cardiac myosin-binding protein C (cMyBP-C), a signalling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of both contraction and relaxation 2 .Current treatment options for HCM provide mostly symptomatic relief without addressing the genetic cause of the disease. Thus, the development of novel strategies to prevent germline transmission of founder mutations is desirable. One approach for preventing second-generation transmission is preimplantation genetic diagnosis (PGD) followed by selection of non-mutant embryos for transfer in the context of an in vitro fertilization (IVF) cycle. When only one parent carries a heterozygous mutation, 50% of the embryos should be mutationfree and available for transfer, while the remaining carrier embryos are discarded. Gene correction would rescue mutant embryos, increase the number of embryos available for transfer and ultimately improve pregnancy rates.Recent developments in precise genome-editing techniques and their successful applications in animal models have provided an option for correcting human germline mutations. In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs 7-10 . DSBs are then resolved by endogenous DNA repair mechanisms, prefer...

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DNA targeting specificity of RNA-guided Cas9 nucleases

Cited by 4,155 publications

References 28 publications

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Correction of a pathogenic gene mutation in human embryos

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