CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.
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.
Department of Cardiothoracic Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.|Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, California 92093-0613, USA.
Xiaolong
X
Ma
Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, California 92093-0613, USA.
Chao
C
Chen
Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, California 92093-0613, USA.
Jennifer
J
Veevers
Department of Cardiothoracic Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
XinMin
X
Zhou
Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, California 92093-0613, USA.|Veterans Administration Healthcare San Diego, 3350 La Jolla Village Drive, San Diego, California 92161, USA.
Robert S
RS
Ross
Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, California 92093-0613, USA.
Wei
W
Feng
Department of Medicine-Cardiology, University of California San Diego, 9500 Gilman Drive, Mail Code 0613-C, La Jolla, California 92093-0613, USA.
Ju
J
Chen
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular; D055786:Gene Knockout Techniques; D016678:Genome; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism
D011494:Protein Kinases