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

• Published in: The FEBS journal 2018-08, Vol.285 (15), p.2828-2839
• Main Authors: Zhang, Ju, Cui, Meng‐Lan, Nie, Yong‐Wei, Dai, Bai, Li, Fei‐Ran, Liu, Dong‐Jun, Liang, Hao, Cang, Ming
• Format: Article
• Language: English
• Subjects: Animal breeding; Animal models; Animals; Animals, Genetically Modified; Cashmere; Cell lines; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Electroporation; Fatty Acid Desaturases - genetics; Fatty Acid Desaturases - metabolism; fat‐1; Female; Fetuses; Fibroblasts; Gene Editing - methods; Gene Knockout Techniques; Genes; Genetic modification; Genetic Vectors; Genomes; Goats; Goats - genetics; Insertion; knockin; knockout; Loci; MSTN; Mutation; Myostatin - genetics; Nuclear transfer; Nuclear Transfer Techniques; Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; Somatic cell nuclear transfer
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14520

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. The CRISPR/Cas9 system has provided a precise and versatile approach for genome editing in various species. Here, simultaneous editing of two genes (simultaneous knockout and knockin) was achieved in Arbas cashmere goat using the CRISPR/Cas9 system. This suggests that the technique has the potential to become an important and applicable gene engineering tool in safe breeding of large mammals.