title: ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.
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.
authors.affiliation: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.|Mouse Genomics Resource Laboratory, National Institute of Genetics, Shizuoka 411-8540, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Yoshimi
authors.affiliation: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.|Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kunihiro
authors.affiliation: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Kaneko
authors.affiliation: BioDynamics Laboratory Inc., Tokyo 113-0033, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nagahora
authors.affiliation: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Birger
authors.initials: B
authors.lastname: Voigt
authors.affiliation: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.|Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
authors.forename: Tomoji
authors.initials: T
authors.lastname: Mashimo
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D005260:Female; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering / Q000379:methods*; D059765:Homologous Recombination / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D051381:Rats; D011971:Receptors, Immunologic / Q000235:genetics; D015053:Zygote / Q000378:metabolism*
chemical_list: D000943:Antigens, Differentiation; D009838:Oligodeoxyribonucleotides; D011971:Receptors, Immunologic; C501920:SIRPA protein, human