Generation of gene-modified mice via Cas9/RNA-mediated gene targeting

Shen, Bin; Zhang, Jun; Wu, Hongya; Wang, Jianying; Ma, Ke; Li, Zheng; Zhang, Xueguang; Zhang, Pumin; Huang, Xingxu

doi:10.1038/cr.2013.46

Cited by 517 publications

(450 citation statements)

References 15 publications

Supporting

Mentioning

439

Contrasting

Unclassified

Order By: Relevance

“…RGENs consist of Cas9, invariable transactivating crRNA (tracrRNA) and target-specific crRNA (or sgRNA that consists of essential portions of tracrRNA and crRNA), and are readily reprogrammed by replacing crRNA (or sgRNA) 7 . We and others have recently used RGENs for genome engineering in bacteria 8 , mammalian cells [9][10][11][12] , animals [13][14][15][16][17][18][19] and plants [20][21][22] .…”

mentioning

confidence: 99%

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

et al. 2014

View full text Add to dashboard Cite

Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/ Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.

show abstract

mentioning

confidence: 99%

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

et al. 2014

View full text Add to dashboard Cite

show abstract

“…For each targeting site, a single-guide (sgRNA) was designed using the rules described by Sapranauskas et al [9] (Supplementary information, Figure S1 and Table S1). The Cas9 mRNA and sgRNA were transcribed by T7 RNA polymerase in vitro as described by Shen et al [10]. A mixture of Cas9 mRNA (25 ng/µl) and sgRNA (10 ng/µl) was pooled with circular donor vectors (4 ng/µl), and microinjected into one-cell stage fertilized eggs of Sprague Dawley (SD) rat (Supplementary information, Table S2).…”

Section: Dear Editormentioning

confidence: 99%

Generating rats with conditional alleles using CRISPR/Cas9

et al. 2013

Self Cite

View full text Add to dashboard Cite

“…Using mammalian cell lines, they demonstrated that indels and knock-ins can be introduced by co-transfection of sgRNA and Cas9 expression vectors with higher efficiency than that of TALENs. The first paper describing CRISPR/Cas9-mediated in vivo genome editing in mouse zygotes was also published in the same year [7,8]. Shen et al [7] showed that the EGFP gene in Pou5f1-IRES-EGFP knock-in mice can be mutated by microinjection of in vitro transcribed sgRNA and Cas9 mRNA into fertilized eggs.…”

Section: Introductionmentioning

confidence: 99%

“…The first paper describing CRISPR/Cas9-mediated in vivo genome editing in mouse zygotes was also published in the same year [7,8]. Shen et al [7] showed that the EGFP gene in Pou5f1-IRES-EGFP knock-in mice can be mutated by microinjection of in vitro transcribed sgRNA and Cas9 mRNA into fertilized eggs. Using this strategy, Wang et al [8] demonstrated that NHEJ-mediated indel mutations can be introduced at multiple endogenous genomic loci in vivo.…”

Section: Introductionmentioning

confidence: 99%

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

Hara

Takada

2017

J Hum Genet

View full text Add to dashboard Cite

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.

show abstract

Generation of gene-modified mice via Cas9/RNA-mediated gene targeting

Cited by 517 publications

References 15 publications

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

Generating rats with conditional alleles using CRISPR/Cas9

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

Contact Info

Product

Resources

About