A Self-restricted CRISPR System to Reduce Off-target Effects

Chen, Yanhao; Liu, Xiaojian; Zhang, Yongxian; Wang, Hui; Ying, Hao; Liu, Mingyao; Li, Dali; Lui, Kathy O.; Ding, Qiurong

doi:10.1038/mt.2016.172

Cited by 72 publications

(54 citation statements)

References 12 publications

(11 reference statements)

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“…While recently reported bone marrow chimera approaches can address some of these problems (7), this strategy remains time consuming and unsuitable for studying genes required for thymic T cell development. Second, persistent Cas9 expression both increases the likelihood of off-target effects (8, 9), and is not suitable for mouse in vivo adoptive transfer approaches, as the immunogenic Cas9 protein can cause cell rejection (10, 11).…”

Section: Introductionmentioning

confidence: 99%

Efficient CRISPR/Cas9 gene ablation in uncultured naïve mouse T cells for in vivo studies

Nüssing

House

Kearney

et al. 2019

Preprint

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AbstractCRISPR/Cas9 technologies have revolutionised our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene deletion strategies in T cells require in vitro stimulation or culture that can both preclude studies of gene function within unmanipulated naïve T cells and can alter subsequent differentiation. Here we demonstrate highly efficient gene deletion within uncultured primary naïve murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knock-out cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naïve state, suggesting that gene deletion occurs independent of T cell activation. This protocol thus expands CRISPR-based probing of gene function beyond models of robust T cell activation, to encompass both naïve T cell homeostasis and models of weak activation, such as tolerance and tumour models.

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Section: Introductionmentioning

confidence: 99%

Efficient CRISPR/Cas9 gene ablation in uncultured naïve mouse T cells for in vivo studies

Nüssing

House

Kearney

et al. 2019

Preprint

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“…In addition, the development of tools, which prevent the sustained expression of CRISPR nucleases, are required to restrain the nuclease expression to the minimal time period required for a sufficient editing of the target gene 45,46 , while limiting the occurrence of off-target mutations. Here we empirically assessed the potential offtargets of our sgRNAs based on an in silico prediction tool.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo

Duchêne

Cherif

Iyombe-Engembe

et al. 2018

Preprint

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Duchenne Muscular Dystrophy (DMD), a severe hereditary disease, affecting 1 boy out of 3500, mainly results from the deletion of one or more exons leading to a reading frame shift of the DMD gene that abrogates dystrophin protein synthesis. We used the Cas9 of Staphylococcus aureus (SaCas9) to edit the human DMD gene.Pairs of sgRNAs were meticulously chosen to induce a genomic deletion to not only restore the reading frame but also produced a dystrophin protein with normally phased spectrin-like repeats. The formation of a dystrophin protein with spectrin-like repeats normally phased is not usually obtained by skipping or by deletion of complete exons. This can however be obtained in rare instances where the exon/intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion are at similar positions in the spectrin-like repeat. We used pairs of sgRNAs, targeting exons 47 and 58 and a normal reading frame was restored in 67 to 86% of the resulting hybrid exons in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. The restoration of the DMD reading frame and restoration of the dystrophin expression was also obtained in vivo in the heart of the del52hDMD/mdx. Our results provide a proof-ofprinciple that SaCas9 could be used to edit the human DMD gene and could be considered for the further development of a therapy for DMD.

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“…62 One potential solution is the inclusion in the vector of an additional guide RNA that targets the Cas9 gene itself, resulting in cleavage of the vector that self-limits the duration of expression. 63 An entirely different solution would be the use of lipid nanoparticles to deliver Cas9 messenger RNA into hepatocytes, relieving the size restriction imposed by AAV, as well as providing Cas9 in a form that is rapidly degraded in cells. 64,65 …”

Section: Next Stepsmentioning

confidence: 99%

CRISPR-Cas9 Genome Editing for Treatment of Atherogenic Dyslipidemia

Chadwick

Musunuru

2018

ATVB

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Although human genetics has resulted in the identification of novel lipid-related genes that can be targeted for the prevention of atherosclerotic vascular disease, medications targeting these genes or their protein products have short-term effects and require frequent administration during the course of the lifetime for maximal benefit. Genome-editing technologies, such as CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR–associated 9) have the potential to permanently alter genes in the body and produce long-term and even lifelong protection against atherosclerosis. In this review, we discuss recent advances in genome-editing technologies and early proof-of-concept studies of somatic in vivo genome editing in mice that highlight the potential of genome editing to target disease-related genes in patients, which would establish a novel therapeutic paradigm for atherosclerosis.

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A Self-restricted CRISPR System to Reduce Off-target Effects

Cited by 72 publications

References 12 publications

Efficient CRISPR/Cas9 gene ablation in uncultured naïve mouse T cells for in vivo studies

Efficient CRISPR/Cas9 gene ablation in uncultured naïve mouse T cells for in vivo studies

CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo

CRISPR-Cas9 Genome Editing for Treatment of Atherogenic Dyslipidemia

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