Gene Targeting with TALEN and CRISPR/Cas9 in Pigs and Beyond

Genome editing technology combined with the somatic cell nuclear transfer (SCNT) method has been widely applied to generate gene knockout pigs. However, the enrichment efficiency of biallelic knockout (KO) cells was still low. Very limited information is known regarding the off-target and the heredity of TALEN or CRISPR-Cas9 modified pigs and offspring. Here, we describe the development method of efficiently enrich mutated somatic cells with TALEN and CRISPR/Cas9, and gene knockout pigs were produced at a highly efficient manner. GGTA1 knockout Wuzhishan and Bama miniature pigs were produced using a combination of TALEN mRNA and magnetic beads with SCNT. A total of 115 cell colonies were formed and validated to be GGTA1 KO cells by PCR and sequencing, and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. 30 GGTA1 biallelic KO piglets were successfully delivered and grew normally. 17 potential off-target sites were investigated, and no off-target events were detected in the live piglets. GGTA1 biallelic KO founders were mated with wild type and the mutations were transmitted to the F1 piglets. Surprisingly, wildtypes were identified from the 5 litters, which did not conform to Mendel's law. Then the whole GGTA1 gene of the founder was sequenced by targeted capture sequencing. 7-kb deletion in one allele was found at the target region, which cannot be identified by sequencing PCR product of cell colonies. We used CRISPR-Cas9 and EGFP plasmid co-transfection into the ear fibroblast cells from Wuzhishan and Bama miniature pigs to induce mutation in the CMAH, B4GALNT2, ZBED6, ASGR1, MECP2. After 2d, single cell were cultured after the flow cytometry. The gene targeting efficiency improved 4-5 times, biallelic KO occupied about half of mutation. Using SCNT, we efficiently produced gene knockout pig lines with expected genotypes. The potential off-target sites were investigated, but no off-target mutations were detected in the all five kinds of pigs. Taken together, the KO CMAH, B4GALNT2, ZBED6, ASGR1, MECP2 pigs are good modes for addressing issues in human medicine or animal production. Developing enrich system of mutated somatic cells by magnetic beads, GFP co-transfection and flow cytometry provide an efficiently approach for generating biallelic KO livestock.