Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells

Chen, Yu; Liu, Yanxia; Ma, Tao; Liu, Kai; Xu, Shaohua; Zhang, Yu; Liu, Honglei; Russa, Marie La; Xie, Min; Ding, Sheng; Qi, Lei S.

doi:10.1016/j.stem.2015.01.003

Cited by 387 publications

(311 citation statements)

References 32 publications

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“…In mammalian cells, inhibition of the NHEJ pathway can increase HR efficiency (Maruyama et al 2015;Yu et al 2015). To determine whether this is also the case in Drosophila, we compared the knockout efficiencies of five lncRNA genes in wild-type and Lig4 −/− mutant flies.…”

Section: Systematic Identification Of Testis-associated Lncrnas In Drmentioning

confidence: 99%

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

et al. 2016

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Long noncoding RNAs (lncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although lncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. In this study, we identified 128 testis-specific Drosophila lncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the lncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans. Furthermore, gene expression profiles for five lncRNA mutants revealed that testis-specific lncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific lncRNAs evolved much faster. Moreover, lncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, our results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis.

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Section: Systematic Identification Of Testis-associated Lncrnas In Drmentioning

confidence: 99%

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

et al. 2016

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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: Discussionmentioning

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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“…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]. These different approaches could be worth exploring to better control CRISPR-assisted genome editing in plants and to progress in the understanding of the cellular mechanisms involved.…”

Section: Understanding and Controlling The Dna Repair Pathways Involvmentioning

confidence: 99%

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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Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells

Cited by 387 publications

References 32 publications

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

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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