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.