Disruption of exogenous eGFP gene using RNA-guided endonuclease in bovine transgenic somatic cells

作者：Choi WooJae; Yum SooYoung; Lee SongJeon; Lee WonWu; Lee JiHyun; Kim SeokJoong; Koo OkJae; Lee ByeongChun; Jang Goo^*

来源：Zygote, 2015, 23(6): 916-923.

DOI：10.1017/S096719941400063X

• CRISPR-Cas9
• eGFP disruption
• PiggyBac
• Transgenic cattle

Genome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6-203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science.

• 出版日期2015-12