Targeted gene disruption in Xenopus laevis using CRISPR/Cas9

Wang, Fengqin; Shi, Zhaoying; Cui, Yan; Guo, Xiaogang; Shi, Yun‐Bo; Chen, Yonglong

doi:10.1186/s13578-015-0006-1

Cited by 100 publications

(87 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CRISPR/Cas9 genome modification is a powerful approach for generating both targeted indel mutations by non-homologous endjoining (NHEJ) repair and more precise gene editing by the introduction of specific sequences through homology-directed repair (HDR) (Auer et al, 2014;Bassett et al, 2014;Bhattacharya et al, 2015;Blitz et al, 2013;Chen et al, 2013;Friedland et al, 2013;Guo et al, 2014;Hisano et al, 2015;Kimura et al, 2014;Kotani et al, 2015;Li et al, 2015;Nakayama et al, 2013Nakayama et al, , 2014Ran et al, 2013;Shi et al, 2015;Wang et al, 2015;Yang et al, 2014). While NHEJ-mediated indel mutations have proven to be effective in animal models including mouse, zebrafish and Xenopus (Blitz et al, 2013;Kotani et al, 2015;Nakayama et al, 2013;Wang et al, 2015;Xue et al, 2014), HDR-mediated targeted DNA insertion has proven more challenging.…”

Section: Introductionmentioning

confidence: 99%

“…While NHEJ-mediated indel mutations have proven to be effective in animal models including mouse, zebrafish and Xenopus (Blitz et al, 2013;Kotani et al, 2015;Nakayama et al, 2013;Wang et al, 2015;Xue et al, 2014), HDR-mediated targeted DNA insertion has proven more challenging. Microhomology-mediated end-joining (MMEJ)-dependent integration has been demonstrated in Xenopus, but germline transmission of the integrated sequence was not observed and F0 embryos are primarily mosaic (Nakade et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High efficiency non-mosaic CRISPR mediated knock-in and mutations in F0 Xenopus

et al. 2017

View full text Add to dashboard Cite

The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High efficiency non-mosaic CRISPR mediated knock-in and mutations in F0 Xenopus

et al. 2017

View full text Add to dashboard Cite

show abstract

“…We and others have shown that programmable nucleases are efficient genome editing tools in the human disease model Xenopus, both in the diploid frog Xenopus tropicalis and its close allotetraploid relative Xenopus laevis (Young et al, 2011;Ishibashi et al, 2012;Lei et al, 2012;Blitz et al, 2013;Nakayama et al, 2013;Guo et al, 2014;Nakajima and Yaoita, 2015a;Wang et al, 2015). In an attempt to circumvent founder lethality, we sought to develop a method to confine targeted gene mutations to the germline, thereby 'protecting' somatic tissues from the deleterious effects of LOF mutations.…”

Section: Introductionmentioning

confidence: 99%

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

2016

View full text Add to dashboard Cite

CRISPR/Cas9 genome editing is revolutionizing genetic loss-offunction analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.

show abstract

“…CRISPR/Cas9 application to the mouse has been discussed by recent protocol and review papers [51-54] so we will not discuss these studies here. Frogs, Xenopus tropicalis and Xenopus laevis, have been the subject of several CRISPR/Cas9 genome editing studies [55][56][57] and for medaka Oryzias latipes there is to date a couple of papers with an initial demonstration of CRISPR/Cas9 applicability [58,59].…”

Section: Crispr/cas9 In Animal Modelsmentioning

confidence: 99%

Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9

Ceasar

Rajan

Prykhozhij

et al. 2016

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

115

View full text Add to dashboard Cite

The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.

show abstract

Targeted gene disruption in Xenopus laevis using CRISPR/Cas9

Cited by 100 publications

References 9 publications

High efficiency non-mosaic CRISPR mediated knock-in and mutations in F0 Xenopus

High efficiency non-mosaic CRISPR mediated knock-in and mutations in F0 Xenopus

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9

Contact Info

Product

Resources

About