RRC ID 35757
Author Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T.
Title ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.
Journal Nat Commun
Abstract The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.
Volume 7
Pages 10431
Published 2016-1-20
DOI 10.1038/ncomms10431
PII ncomms10431
PMID 26786405
PMC PMC4736110
MeSH Animals Antigens, Differentiation / genetics CRISPR-Cas Systems / genetics* Clustered Regularly Interspaced Short Palindromic Repeats / genetics Female Gene Knock-In Techniques Genetic Engineering / methods* Homologous Recombination / genetics Humans Male Mice Oligodeoxyribonucleotides / genetics* Rats Receptors, Immunologic / genetics Zygote / metabolism*
IF 12.121
DNA material T7-NLS hCas9-pA (RDB13130) pCAGGS (RDB08938)