Abstract：
:Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Ma X,Chen C,Veevers J,Zhou X,Ross RS,Feng W,Chen Jdoi
10.1038/srep42244subject
Has Abstractpub_date
2017-02-08 00:00:00pages
42244issn
2045-2322pii
srep42244journal_volume
7pub_type
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