SsODN-mediated Knock-in with CRISPR-Cas for Large Genomic Regions in Zygotes

Overview

Journal Nat Commun

Specialty Biology

Date 2016 Jan 21

PMID 26786405

Citations 172

Authors

Kazuto Yoshimi

Yayoi Kunihiro

Takehito Kaneko

Hitoshi Nagahora

Birger Voigt

Tomoji Mashimo

Affiliations

Soon will be listed here.

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.

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