Figure 4.

CRISPR/Cas9 using dual sgRNAs can effectively knockout PDX1 in sheep. (a) Schematic representation of the two gRNAs designed to target PDX1 loci in sheep. Dual sgRNA microinjection can induce a bi-allelic deletion that can be identified by PCR amplification and gel electrophoresis of the target region. The full-length gel is presented in the Supplementary Information. Mutation efficiency of the dual sgRNA microinjection is presented in the pie chart. From 21 microinjected oocytes 4 had mono-allelic and 4 had bi-allelic deletions. (b) Two sgRNAs targeting PDX1 gene were microinjected into the MII II oocytes before IVF, cultured in vitro for 6 days and transfer to a recipient sheep. The fetus was collected at 4 months of gestation. (c) Genomic DNA was isolated and subjected to PCR, sub-cloning and Sanger sequencing. All of the sequenced colonies showed mutations with a 208 bp deletion. (d) Gel electrophoresis of PCR product -using specific primers for PDX1- from different tissues (liver, lung, heart, kidney, muscle and spleen) of the mutant fetus. Full-length gel is presented in the Supplementary information. (e) Protein sequence of the disrupted allele of the PDX1-KO sheep fetus is shown in red.