Efficient generation of goats with defined point mutation (I397V) in GDF9 through CRISPR/Cas9

The recent emergence of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has attracted significant attention for its potential to improve traits of agricultural importance. However, most applications in livestock species to date have depended on aberrant DNA repair to generate frameshifting indels. Whether this genomic engineering technique involving homology-dependent repair (HDR) can be used to introduce defined point mutations has been less explored. Previously, we reported a G→A point mutation (g.231A>G, p.Val397Ile) in the growth differentiation factor 9 (GDF9) gene that has a large effect on the litter size of cashmere goats. In the present study we report that by co-injecting synthesised RNAs and single-stranded oligo deoxynucleotide (ssODN) donor sequences into goat zygotes, we successfully introduced defined point mutations resulting in single amino acid substitutions in the proteins as expected. The efficiency of this precise single-nucleotide substitution in newborn kids was as high as 24% (4/17), indicating that ssODN-directed HDR via zygote injection is efficient at introducing point mutations in the goat genome. The findings of the present study further highlight the complex genome modifications facilitated by the CRISPR/Cas9 system, which is able to introduce defined point mutations. This represents a significant development for the improvement of reproduction traits in goats, as well as for validating the roles of specific nucleotides in functional genetic elements in large animals.