Genome Editing in Rats via CRISPR-Cas

Genome editing by CRISPR-Cas was performed as described previously (Yoshimi et al., 2016 (link)). Guide RNAs (gRNAs) were designed by Optimized CRISPR Design (crispr.mit.edu) and synthesized by Integrated DNA Technologies, Inc., (Coralville, IA, United States). Long ssODNs (lsODNs) were prepared using a LsODN Preparation Kit (Biodynamics Laboratory Inc., Tokyo, Japan). Cas9 protein was purchased from Integrated DNA Technologies. Pronuclear-stage embryos of F344/Jcl (CLEA Japan, Inc., Tokyo, Japan) rats were produced by natural mating. The oviducts of female rats with vaginal plugs were removed after euthanasia by CO₂ and cervical dislocation, and embryos were flushed out from the ampullae with culture medium. Cas9 protein, gRNAs, and lsODN were introduced into the embryos using a super electroporator NEPA 21 (NEPA GENE Co., Ltd., Ichikawa, Chiba, Japan). Embryos that developed to the two-cell stage were transferred into the oviducts of pseudopregnant females that were anesthetized using isoflurane. Offspring were genotyped by the Amp-FTA method with the following primer set: 5′-CGGGTTTCAGAGATGGAAGA-3′ and 5′-ATTTTCATTGACAGGTCCGG-3′ and Ampdirect Plus buffer (Shimadzu Corporation, Kyoto, Japan). Founder rats were mated with F344/Jcl rats and F1 heterozygous rats were intercrossed to obtain F2 progeny.