Figure 2. Detection of Npc1l1sgRNA:Cas9-mediated modifications of NPC1L1 in founder pigs.

(a) A photo showing 38-day-old pigs carrying Npc1l1 mutations. (b) PCR products of the targeted region of Npc1l1 from founder pigs. The first litter of five live pigs were named C1-1 to C1-5. The second litter of six live pigs were named from C2-1 to C2-6. Con denotes wild-type pig as a control. Notably, the PCR products of C1-3, C1-4, C2-1, C2-4 and C2-6 differ from the control in size, suggesting a large fragment deletion or insertion. (c) Detection of Npc1l1 sgRNA:Cas9-mediated on-target cleavage of Npc1l1 by T7EN1 cleavage assays. All PCR products from (b) were subjected to T7EN1 cleavage assays. All the samples were digested by T7EN1, suggesting that all founders carry Npc1l1 mutations. (d) Sequencing results of modified Npc1l1 alleles detected in founder pigs. At least 12 TA clones of the PCR products were analyzed. Sequences complementary to sgRNA are labeled in red, and PAM sequences in green. Mutations, blue, lower case; deletions, (−); insertions, (+). N/N indicates positive colonies out of total sequenced samples. See also Figure S1.