MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems

Sakuma, Tetsushi; Nakade, Shota; Sakane, Yuto; Suzuki, Ken; Yamamoto, Takashi

doi:10.1038/nprot.2015.140

Cited by 333 publications

(314 citation statements)

References 49 publications

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“…However, for most clinical treatments or precise modeling of diseases in vitro, it is essential to achieve high-frequency knock-in of the repair template with the desired variant. 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].…”

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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Section: Discussionmentioning

confidence: 99%

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

2017

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“…However, the discovery that CRISPR Cpf1/gRNA generates a DSB with a 5′ overhang suggests that Obligare may be feasible using this newly identified RNA-guided endonuclease . The use of microhomology-mediated end-joining (MMEJ) techniques such as PITCh may also be feasible with TALENs and Cas9/gRNA (Sakuma et al 2016).…”

Section: Are There Still Better Ways To Edit?mentioning

confidence: 99%

Impact of gene editing on the study of cystic fibrosis

2016

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Cystic fibrosis (CF) is a chronic and progressive autosomal recessive disorder of secretory epithelial cells, which causes obstructions in the lung airways and pancreatic ducts of 70,000 people worldwide (for recent review see Cutting Nat Rev Genet 16(1):45-56, 2015). The finding that mutations in the CFTR gene cause CF (Kerem et al. Science 245(4922):1073-1080, 1989; Riordan et al. Science 245(4922):1066-1073, 1989; Rommens et al. Science 245(4922):1059-1065, 1989), was hailed as the very happy middle of a story whose end is a cure for a fatal disease (Koshland Science 245(4922):1029, 1989). However, despite two licensed drugs (Ramsey et al. N Engl J Med 365(18):1663-1672, 2011; Wainwright et al. N Engl J Med 373(3):220-231, 2015), and a formal demonstration that repeated administration of CFTR cDNA to patients is safe and effects a modest but significant stabilisation of disease (Alton et al. Lancet Respir Med 3(9):684-691, 2015), we are still a long way from a cure, with many patients taking over 100 tablets per day, and a mean age at death of 28 years. The aim of this review is to discuss the impact on the study of CF of gene-editing techniques as they have developed over the last 30 years, up to and including the possibility of editing as a therapeutic approach.

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“…In human cells, a direct correlation was observed between the efficiency of HDR of CRISPR-induced DSBs and the size of the homology arms of the donor template, better knock-in efficiency being obtained with homology regions of at least 1 kb [65]. However, in the MMEJ (microhomology-mediated end joining)-mediated method of gene knock-in named PITCh (precise integration into target chromosome), very short fragments (5-25 bp) homologous to sequences framing the target are used to trigger the insertion of the donor template via MMEJ and not HR [66].…”

Section: With Homology To the Targetmentioning

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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MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems

Cited by 333 publications

References 49 publications

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

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

Impact of gene editing on the study of cystic fibrosis

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

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