Sequence analysis of blastocysts injected with all-in-one FokI-dCas9 vector simultaneously targeting Reg3b and Reg3g genes. The PCR products were analyzed by direct sequencing to identify mutations in Reg3b
(A) and Reg3g
(B) loci. The wild-type sequence of Reg3b and Reg3g are shown at the top (Wild) with the gRNA target sequences (underlined). The PAM sequence is enclosed in red boxes. Deletions are indicated by dashes. Blastocyst numbers on the left side of each sequence in (A) and (B) are identical. Most blastocysts had multiple types of mutations at the Reg3b locus. The wild-type Reg3b sequence was not detected in blastocyst #4 and #6. In the other blastocysts, the wild-type Reg3b sequence was detected along with mutant sequence. Regarding Reg3g locus, the wild-type sequence was detected in all blastocysts with mutant sequence.