19 CRISPR/Cas9-based gene editing for generation of cloned goat embryos with enhanced pashmina fiber-producing potential

The objective of this study was to generate fibroblast growth factor-5 (FGF5) gene-edited changthangi (pashmina) goat embryos. We generated a dual guide RNA (gRNA)-bearing plasmid construct harboring the DNA sequence of the Cas9 enzyme and two gRNAs targeting the knockout of a part (259 bp) of exon 1 of the FGF5 gene. The gRNAs were designed using the CRISPOR (crispor.gi.ucsc.edu) online tool. Each gRNA sequence was cloned into a linearized PX459 vector backbone to generate two plasmid constructs. The two constructs were assembled by the Gibson cloning method. Validation of the final plasmid was done by Sanger sequencing, and the plasmid integrity was checked on a 1% agarose gel. Transfection of editing constructs into pashmina goat dermal fibroblasts was performed using the NEPA21 Superelectroporator at passages 3–4 and 90%–100% cell confluency. After PCR confirmation of signified editing events, the single-cell pickup method was used for the generation of single-cell clones in 96-well plates. The cell clones that revealed bi-allelic deletions on PCR detection were used as somatic cell donors. Cloned reconstructs or doublets (enucleated oocyte + single cell) were generated using a modified zona-free nuclear transfer technique. The doublets generated were incubated for 3–4 h to allow for reprogramming of the donor nuclear DNA by the ooplasmic reprogramming factors. After complete fusion and rounding-off, the reconstructs were chemically activated using calcium ionophore and 6-dimethylaminopurine (6-DMAP) protocol. After the 6-DMAP treatment, the cloned embryos were in vitro cultured in the WID (Well in Drop) culture system for blastocyst formation. BO medium was used as the in vitro embryo culture (IVC) medium in all experiments. Embryo development was assessed on Day 7 (after 140 h) after IVC. We successfully produced bi-allelic FGF5 gene-edited cell lines with excision of 259 bp in exon 1 of the FGF5 gene at the expected location. The editing events in the dermal fibroblasts were confirmed by Sanger sequencing. Furthermore, we also developed a novel PCR-based detection method for confirmation of editing events. The targeting efficiency of FGF5 in cultured primary fibroblasts was as high as 59.1%, while the efficiency of disrupting FGF5 in 31 tested single-cell clones was 21.03% for heterogeneous and 12.9% for homozygous gene modifications. The blastocyst percentage using nonedited cells and edited cells of same lineage was 13.4% and 11.3%, respectively. In accordance with the sequencing results, editing at specific sites could be accomplished. This outcome suggests that the CRISPR/Cas9 system will be crucial for precision breeding because it has the potential to develop into a reliable and effective tool for gene engineering in farm animals.