Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats.

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN-/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.

CRISPR/Cas9-mediated specific integration of fat-1 at the goat MSTN locus.

Recent advances in understanding the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, no study has reported simultaneous knockout of endogenous genes and site-specific knockin of exogenous genes in large animal models. Using the CRISPR/Cas9 system, this study specifically inserted the fat-1 gene into the goat MSTN locus, thereby achieving simultaneous fat-1 insertion and MSTN mutation. We introduced the Cas9, MSTN knockout small guide RNA and fat-1 knockin vectors into goat fetal fibroblasts by electroporation, and obtained a total of 156 positive clonal cell lines. PCR and sequencing were performed for identification. Of the 156 clonal strains, 40 (25.6%) had simultaneous MSTN knockout and fat-1 insertion at the MSTN locus without drug selection, and 55 (35.25%) and 101 (67.3%) had MSTN mutations and fat-1 insertions, respectively. We generated a site-specific knockin Arbas cashmere goat model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer for the first time. For biosafety, we mainly focused on unmarked and non-resistant gene screening, and point-specific gene editing. The results showed that simultaneous editing of the two genes (simultaneous knockout and knockin) was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in safe animal breeding.