Myotube differentiation in clustered regularly interspaced short palindromic repeat/Cas9-mediated MyoD knockout quail myoblast cells.
OBJECTIVE
In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells.


METHODS
TheMyoDgene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation.


RESULTS
The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4) through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation.


CONCLUSIONS
With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
Si Won
SW
Kim
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
Jeong Hyo
JH
Lee
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
Byung-Chul
BC
Park
Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
Tae Sub
TS
Park
CRISPR-Cas9; Knockout; Muscle Differentiation; MyoD; Myoblast