RRC ID |
35757
|
著者 |
Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T.
|
タイトル |
ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.
|
ジャーナル |
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.
|
巻・号 |
7
|
ページ |
10431
|
公開日 |
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
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
リソース情報 |
遺伝子材料 |
T7-NLS hCas9-pA (RDB13130)
pCAGGS (RDB08938) |