earticle

영어

To date, CRISPR/CRISPR-associated protein 9 (Cas9)-mediated genome editing technology is expected to become the most efficient tool for economic trait improvement in livestock. However, one of the critical issues for genetic modification by CRISPR/Cas9 is non-specific DNA double strand breaks which is called as off-target effect. In this study, we examined the mutated Cas9, nickase to modulate the specific target gene in chicken primordial germ cells (PGCs) as well as DF1 cells. Chicken myostatin which is famous for inhibitor of muscle cell growth and differentiation during myogenesis was targeted to be mutated with the Cas9-D10A nickase. After co-transfection of the Cas9- D10A nickase expression vector with eGFP gene and two targeted guide RNAs (g- RNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting (FACS). In DF1 cells which is chicken embryonic fibroblast cell line, the mutant induction efficiency was 100% in the targeted myostatin site by the genotyping analysis of the knockout DF1 cells. In DF1 cells, number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. In addition, 6 expected off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. We also verified myostatin gene knockout with Cas9-D10A nickase in chicken primordial germ cells (PGCs). In conclusion, the knockout technical platform with the Cas9- D10A nickase can be efficiently applied to functional genomics study in poultry and finally adapted to generate the knockout poultry.