Highly efficient enrichment of porcine cells with deletions induced by CRISPR/Cas9 using dual fluorescence selection.

Production of nuclear donor cells with a high percentage of desired modifications is a critical step in the successful generation of genetically modified pigs through somatic cell nuclear transfer (SCNT). The CRISPR/Cas9 system has been used for efficient modification of the nuclear DNA in eukaryotic cells, including porcine cells. However, in vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their enrichment. Here we investigate a dual fluorescence selection system for the efficient enrichment of porcine embryonic fibroblasts (PEFs) with CRISPR/Cas9-induced chromosomal deletions. Enrichment of cells with 170 bp deletions reached a frequency of 74%, whilst enrichment of cells with a larger 5 kb deletions achieved a frequency of 46%. This demonstrates the utility of a dual fluorescence reporter as an attractive tool for improving the efficiency of generating genome edited pigs.