RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

Song, Jun; Yang, Dongshan; Xu, Jie; Zhu, Tianqing; Chen, Y. Eugene; Zhang, Jifeng

doi:10.1038/ncomms10548

Cited by 349 publications

(330 citation statements)

References 28 publications

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“…Another way to improve the efficiency is enhancement of the HDR pathway. Song et al [16] showed that overexpression of RAD51 in zygotes, an essential player in the HDR pathway, or treatment with a RAD51 enhancer, RS-1, also improved HDR-mediated in vivo knock-in efficiencies in rabbits [17].…”

Section: Hdr-mediated Production Of Nucleotide Substitution In Micementioning

confidence: 99%

Genome editing for the reproduction and remedy of human diseases in mice

Hara

Takada

2017

J Hum Genet

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With the recent progress in genome-editing technologies, such as the CRISPR/Cas9 system, genetically modified animals carrying nucleotide substitutions or large chromosomal rearrangements can be produced rapidly and at low cost. Such genome-editing techniques have been applied in the generation of animal models, especially mice, for reproducing human disease mutations, such as single-nucleotide polymorphisms (SNPs) or large chromosomal rearrangements identified by genome-wide screening analyses. While application methods are under development for various complex mutations involving genome editing for mimicking human disease-causing mutations in mice, functional studies of mouse models carrying replicated human mutations are gradually being published. In this review, we discuss the recent progress in application methods of the CRISPR/Cas9 system, focusing on the production of mouse models of diseases.

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Section: Hdr-mediated Production Of Nucleotide Substitution In Micementioning

confidence: 99%

Genome editing for the reproduction and remedy of human diseases in mice

Hara

Takada

2017

J Hum Genet

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“…Notably, since microhomology-mediated end joining (MMEJ)-assisted gene knock-in named PITCh (Precise Integration into Target Chromosome) [103,104] and NHEJ-mediated sitespecific gene insertion, HITI [68], are both HDRindependent precise knock-in methods, these strategies may increase the utility of genome editing in human cultured cells. Other techniques to enhance gene knock-in include inhibition of NHEJ with small compounds or Cas9 protein accumulation in an S-and G 2 -phase-dependent manner [105][106][107][108][109]. Several techniques to shift the DSB repair balance from NHEJ to HDR in human cultured cells have been developed.…”

Section: Resultsmentioning

confidence: 99%

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

2017

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Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.

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“…Higher rates of homology-driven knock-in were also observed in mammalian cells when using hCas9 fused to proteins involved in cell-cycle dynamics [101,102], allowing a low expression of the nuclease in G1 and a high expression during S/G2/M phases where HDR mediated repair is favored [103,104]. In mammalian cells, CRISPR-induced knock-in efficiency was improved when applying inhibitors of the NHEJ pathway, such as Scr7 (inhibiting ligaseIV) [105] or enhancers of the HDR pathway, like RS-1 [106]. A method for high-throughput identification of small chemical compounds capable of enhancing CRISPR-mediated HDR efficiency by 3-fold for large insertions and 9-fold for gene replacement was developed in mammalian cells [107].…”

Section: Understanding and Controlling The Dna Repair Pathways Involvmentioning

confidence: 98%

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Collonnier

Guyon‐Debast

Maclot

et al. 2017

Methods

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a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

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RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

Cited by 349 publications

References 28 publications

Genome editing for the reproduction and remedy of human diseases in mice

Genome editing for the reproduction and remedy of human diseases in mice

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

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