Abstract
The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.
Highlights
Existence of a PAM sequence (-NGG for Cas[9] from S. pyogenes) directly downstream of the seed region of the target site[12,13]
Through injection of Cas[9] protein, we generated mouse strains with large genomic DNA deletions as well as mouse or rat strains with site-specific insertion of Cre or reporter cassettes
Through large DNA fragment deletion by Cas9/single guide RNA (sgRNA), it is practicable to generate mouse strains to model human genetic disorders which are caused by the loss of large DNA fragments
Summary
Existence of a PAM (protospacer-adjacent motif) sequence (-NGG for Cas[9] from S. pyogenes) directly downstream of the seed region of the target site[12,13]. Two groups reported that through introduction of recombinant Cas[9] protein and sgRNAs into cells, both the activity and the accuracy of the CRISPR/Cas system has been improved[17,18]. Initial work suggested highly efficient generation of gene knockout zebrafish and mice through direct injection of Cas[9] protein/sgRNA19,20, only a few reports described the successful knockout of a large DNA fragment[21] or knock-in of a functional gene cassette using this method[22,23,24]. We investigated the generation of multiple animal models using Cas[9] recombinant protein for large genomic DNA deletion and functional gene cassette knock-in. Our study suggests that recombinant Cas[9] protein is an excellent alternative choice for generation of various genetically modified animal models
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