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

Kazuto Yoshimi, Yayoi Kunihiro, Takehito Kaneko, Hitoshi Nagahora, Birger Voigt and Tomoji Mashimo ()
Additional contact information
Kazuto Yoshimi: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
Yayoi Kunihiro: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
Takehito Kaneko: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
Hitoshi Nagahora: BioDynamics Laboratory Inc.
Birger Voigt: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
Tomoji Mashimo: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University

Nature Communications, 2016, vol. 7, issue 1, 1-10

Abstract: 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.

Date: 2016
References: Add references at CitEc
Citations: View citations in EconPapers (1)

Downloads: (external link)
https://www.nature.com/articles/ncomms10431 Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms10431

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/ncomms10431

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().