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.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is a powerful tool for generating genetically modified animals; targeted knock-in (KI) via homologous recombination remains difficult in zygotes
Single-stranded oligodeoxynucleotides have been used as donor templates in combination with the engineered nucleases for efficient targeted insertion of small DNA fragments. single-stranded oligodeoxynucleotides (ssODNs)-mediated KI in mammalian cells occurs via homology-directed repair (HDR) and is more efficient than using double-stranded donor plasmids[18,19,20,21]
Eggs treated with Cas9-green fluorescent protein (GFP)-poly(A) (n 1⁄4 23) showed significantly higher fluorescence intensity compared with eggs injected with Cas9-GFP without poly(A) (n 1⁄4 20) (Fig. 1b,c)
Summary
The CRISPR-Cas system is a powerful tool for generating genetically modified animals; targeted knock-in (KI) via homologous recombination remains difficult in zygotes. 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, simplifying genome engineering in living organisms. Targeted knock-in (KI) can be engineered via homologous recombination (HR) by co-injection of donor plasmids, including large DNA fragments such as green fluorescent protein (GFP) cassettes, with any of the above mentioned nucleases[10,11,12,13,14,15] This was traditionally achieved by constructing targeting vectors with two homology arms of 0.5–1 kb on either side of the insert DNA. The significant technical advantage of this ssODN-mediated end joining approach is its simplicity, as there is no need to construct homology arms in the donor vector, which is especially difficult to perform in larger BACs
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