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

• Published in: Nature communications Vol. 7; no. 1; p. 10431
• Main Authors: Yoshimi, Kazuto, Kunihiro, Yayoi, Kaneko, Takehito, Nagahora, Hitoshi, Voigt, Birger, Mashimo, Tomoji
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 20-01-2016; Nature Publishing Group; Nature Portfolio
• Subjects: 42/41; 42/70; 631/1647/1511; 631/1647/1513/1967/3196; 631/208; 631/337; 64; 64/110; Albinism; Animals; Antigens, Differentiation - genetics; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Cas Systems - genetics; Female; Gene Knock-In Techniques; Genetic Engineering - methods; Genomes; Homologous Recombination - genetics; Humanities and Social Sciences; Humans; Laboratory animals; Male; Mice; multidisciplinary; Mutation; Oligodeoxyribonucleotides - genetics; Plasmids; Proteins; Rats; Receptors, Immunologic - genetics; Science; Science (multidisciplinary); Zygote - metabolism; Japan
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms10431

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.
CRISPR-Cas9 is a powerful genome engineering tool but gene knock-in is limited by fragment size and efficiency of recombination. Here the authors used a modified strategy employing single-strand oligonucleotides to efficiently knock-in large DNA fragments and humanise native rat loci.