Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

Wang, Liren; Shao, Yanjiao; Guan, Yuting; Li, Liang; Wu, Lijuan; Chen, Fangrui; Liu, Meizhen; Chen, Huaqing; Ma, Yanlin; Ma, Xueyun; Liu, Mingyao; Li, Dali

doi:10.1038/srep17517

Cited by 86 publications

(57 citation statements)

References 43 publications

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“…Wang et al [30] reported a 95 kb deletion in mice. Recently, we demonstrated generation of mice carrying deletions of 0.5, 2, and 5 Mb and an inversion of 5 Mb [31,32].…”

Section: Reproduction Of Diseases In Mice Carrying Chromosome Rearranmentioning

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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“…Wang et al [30] reported a 95 kb deletion in mice. Recently, we demonstrated generation of mice carrying deletions of 0.5, 2, and 5 Mb and an inversion of 5 Mb [31,32].…”

Section: Reproduction Of Diseases In Mice Carrying Chromosome Rearranmentioning

confidence: 99%

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

Hara

Takada

2017

J Hum Genet

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“…Therefore, this system can be used to generate genetically modified mice rapidly by microinjecting sgRNAs with Cas9 into fertilized eggs [4,5,6,7,8,9]. Several studies have reported the induction of an efficient CRISPR/Cas9-mediated genomic rearrangement such as deletion or inversion of ~100 kb [10,11,12]. However, the maximum size of indels that can be induced by the CRISPR/Cas9 system is still unclear.…”

mentioning

confidence: 99%

Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

Hara

Kato

Goto

et al. 2016

Journal of Reproduction and Development

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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for genome editing. In this study, using a microinjection-based CRISPR/Cas9 system, we efficiently generated mouse lines carrying mutations at the Irx3 and Irx5 loci, which are located in close proximity on a chromosome and are functionally redundant. During the generation of Irx3/Irx5 double mutant mice, a deletion of ~0.5 Mb between the Irx3 and Irx5 loci was unintentionally identified in 6 out of 27 living pups by PCR based genotyping analysis. This deletion was confirmed by DNA fluorescence in situ hybridization analysis of fibroblasts. These results indicate that the mutant mice with a deletion of at least 0.5 Mb in their genome can be generated by the CRISPR/Cas9 system through microinjection into fertilized eggs. Our findings expand the utility of the CRISPR/Cas9 system in production of disease model animals with large deletions.

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“…Although the ribonuceoprotein complexes of recombinant Cas9 protein combined with synthetic guide RNAs have been demonstrated as effective genome editing tool [12][13][14], their efficiency is still much lower than the conventional plasmid transfer approach. Thus, the plasmid based approach would be the mostly used method in the coming years and scientists working on improving the efficiency of custom guide RNA cloning and IVT (in vitro transcription) synthesis will appreciate the strategy employed here.…”

Section: Discussionmentioning

confidence: 99%

Optimized Plasmid Construction Strategy for Cas9

Li²,

Hossen

et al. 2018

Cell Physiol Biochem

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Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.

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Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

Cited by 86 publications

References 43 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

Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

Optimized Plasmid Construction Strategy for Cas9

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