Fig 1: Evaluation of genome editing efficiency in marmoset early-stage embryos(A) A summary table of early-stage embryo experiments and KI efficiencies under eight conditions (A–H). ∗, the volume was calculated by the size of the injected droplet diameter. ∗∗, in the 2-step injection, donor DNA (100 ng/μL) was injected into the pronucleus, and then RAD51, Cas9, and crRNA+ tracrRNA (50 ng/μL) were injected into the cytoplasm. ∗∗∗, not done. ca., circa.(B) A schematic of the 2-step PCR analysis for KI allele detection (5′ side). In both PCRs, we used 5′-external primers to not amplify the KI vector itself.(C) A representative image of the result of genotyping PCR analysis using amplified genomic DNA (gDNA) from marmoset early-stage embryos. TH-2A-Cre KI was detected by the KI-specific 1.1-kb DNA bands. In this experiment (condition E), 9 of 16 embryos were considered KI positive.(D) A schematic of 1-step PCR analysis for KI allele detection (3′ side). We used 5′-internal and 3′-external primers to amplify only the KI allele.(E) A representative image of the result of genotyping PCR analysis (3′ side) using amplified gDNA from marmoset early-stage embryos. In this experiment (condition E; same samples as used in C), #10 was considered knocked in.(F) DNA sequencing analysis of the KI allele of #10 (3′ side).(G) A schematic of the 2-step PCR analysis for WT allele detection. In the first PCR, we used 5′- and 3′-external primers to not amplify the KI vector and KI allele.(H) A representative image of the result of genotyping PCR (WT allele) analysis using amplified gDNA from marmoset early-stage embryos. In this experiment (condition E; same samples as used in C), all embryos were considered to harbor THWT allele(s).(I) Representative images of DNA sequencing analysis of the THWT allele. As described in the main text, ∼20% (Cas9-DN1S) and ∼30% (Cas9-WT) of embryos harbored indels or large deletion allele(s). Details of each condition are shown in (A).