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Nat Commun. 2016 Jan 20;7:10431. doi: 10.1038/ncomms10431.

ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.

Author information

1
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
2
Mouse Genomics Resource Laboratory, National Institute of Genetics, Shizuoka 411-8540, Japan.
3
Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
4
BioDynamics Laboratory Inc., Tokyo 113-0033, Japan.

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.

PMID:
26786405
PMCID:
PMC4736110
DOI:
10.1038/ncomms10431
[Indexed for MEDLINE]
Free PMC Article

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