Table 2.

Comparison of Cas9–HE and Cas9 in obtaining targeted transgene integration into the rat Rosa26 and Il22bp loci

Cas9 form	Target locus	KI fragments	Dose Cas9 mRNA/sgRNA/donor DNA (ng/µl)	Eggs injected (survival rate %)	Eggs transferred	E14 embryos (% transferred)	Indels + (% of E14)	Donor integration+ (% of E14)	HDR+ (% of E14)
Cas9–HE	Rosa26	CAG-GFP	50/10/2	216 (75%)	154	37 (24%)	29 (78%)	5 (13%)	3 (8%)
Cas9	Rosa26	CAG-GFP	50/10/2	284 (77%)	211	84 (39%)	62 (73%)	2 (2%)	1 (1%)
Cas9–HE	Il22bp	Il22bp-2A-GFP	50/10/2	277 (78%)	217	82 (37%)	45 (55%)	7 (8%)	2 (2%)
Cas9	Il22bp	Il22bp-2A-GFP	50/10/2	286 (80%)	226	91 (40%)	55 (60%)	8 (8%)	4 (4%)

HDR+, HDR-mediated GFP transgene integration was defined as positive in E14 embryos for which both 5′ and 3′ in–out PCRs were detected using primers in the transgene and outside the homology arms (with PCR products confirmed by DNA sequencing, schematic of PCR primer positions in Supplementary Fig. 5); indels+, E14 embryos in which indels were detected by sequencing of PCR amplicons from the targeted genomic region; donor integration, E14 embryos in which PCR amplification of the transgene was positive