Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease

Cho, Seung Woo; Kim, Sojung; Kim, Jong Min; Kim, Jin-Soo

doi:10.1038/nbt.2507

Cited by 1,644 publications

(1,163 citation statements)

References 17 publications

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“…However, CRISPR/Cas9 system just needs synthesized 20‐base‐long sequences to get specificities instead of demanding engineered proteins 24. Besides, CRISPR/Cas9 system has been reported to get higher sequence specificity and targeting efficiency than ZFNs and TALENs 24, 25. Finally, CRISPR/Cas9 system can target multiple sites and induce multiple effects simultaneously, which ZFNs and TALENs lack.…”

Section: Resultsmentioning

confidence: 99%

Exosome–Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs

et al. 2018

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Targeted delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system to the receptor cells is essential for in vivo gene editing. Exosomes are intensively studied as a promising targeted drug delivery carrier recently, while limited by their low efficiency in encapsulating of large nucleic acids. Here, a kind of hybrid exosomes with liposomes is developed via simple incubation. Different from the original exosomes, the resultant hybrid nanoparticles efficiently encapsulate large plasmids, including the CRISPR–Cas9 expression vectors, similarly as the liposomes. Moreover, the resultant hybrid nanoparticles can be endocytosed by and express the encapsulated genes in the mesenchymal stem cells (MSCs), which cannot be transfected by the liposome alone. Taken together, the exosome–liposome hybrid nanoparticles can deliver CRISPR–Cas9 system in MSCs and thus be promising in in vivo gene manipulation.

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Section: Resultsmentioning

confidence: 99%

Exosome–Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs

et al. 2018

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“…Skin fibroblast cultures were expanded and used to generate heterozygous patient induced pluripotent stem cells (iPSCs) as described previously 15 . Two single-guide RNA (sgRNA)-Cas9 [16][17][18] constructs were designed to target this specific MYBPC3 ∆GAGT deletion (Extended Data Fig. 1a, b) along with two exogenous single-stranded oligodeoxynucleotide (ssODN) templates encoding homology arms to the targeted region (Extended Data Table 1).…”

Section: Subject With a Heterozygous Mybpc3 ∆Gagt Deletionmentioning

confidence: 99%

Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

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788

510

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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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“…Within CD34 + HSCs, HDR into the CCR5 locus resulted in 14% modification offering the possibility of targeted gene insertion of C46, an HIV fusion inhibitor than can confer dual protection against R5 and X4-tropic HIV-1 [117]. Targeting of the CCR5 locus using CRISPR/Cas9 has also yielded robust HIV resistance, with mutation efficiencies ranging from 18 to 74.1% [118–120]. …”

Section: Applicationsmentioning

confidence: 99%

Genome-Edited T Cell Therapies

Delhove

Qasim

2017

Curr Stem Cell Rep

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Purpose of ReviewAlternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies.Recent FindingsGenetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors.SummaryThe combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.

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Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease

Cited by 1,644 publications

References 17 publications

Exosome–Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs

Exosome–Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs

Correction of a pathogenic gene mutation in human embryos

Genome-Edited T Cell Therapies

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