Myotube differentiation in clustered regularly interspaced short palindromic repeat/Cas9-mediated MyoD knockout quail myoblast cells

Abstract

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: The MyoD gene 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.

Conclusion: 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.

Keywords: CRISPR-Cas9; Knockout; Muscle Differentiation; MyoD; Myoblast.

Figure 1

MyoD gene knockout in QM7 cells. (A) Genomic sequence and structure of the quail MyoD gene. (B) Expression vector of Cas9-GFP and MyoD guide RNA (gRNA). The U6 promoter controls gRNA transcription followed by a termination signal. (C) Experimental overview and fluorescence-activated cell sorting (FACS) for the development of single cell-derived MyoD knockout QM7 sublines.

Figure 2

Genotyping analysis of MyoD gene knockout QM7 cells. (A) Genotypes of MyoD-knockout mixed QM7 cells. (B) Genotypes of single cell-derived MyoD-knockout QM7 sublines (MyoD KO QM7#4).

Figure 3

Characterization of MyoD-knockout QM7 cells. (A) Paired box 7 (Pax7) expression in regular QM7 (rQM7) and MyoD-knockout QM7 (MyoD KO QM7#4) cells. There were no significant differences between rQM7 and MyoD KO QM7#4 cells. (B) Morphology of undifferentiated rQM7 and MyoD KO QM7#4 cells. (C) Morphological changes 3 or 6 days after differentiation. rQM7 cells transformed into myotubes during differentiation. No morphological changes were evident in MyoD KO QM7#4 cells after 6 days of differentiation. All images were observed under the inverted microscope, scale bars = 200 μm.

Figure 4

(A) Nuclear fusion in regular QM7 (rQM7) and MyoD-knockout QM7 (MyoD KO QM7#4) cells during myotube differentiation. No fusion was evident in MyoD KO QM7#4 cells even after 6 days of differentiation. (B) Western blotting detected MyoD protein in rQM7 and MyoD KO QM7#4 cells after 3 days of differentiation. (C) RT-PCR analysis of myogenic differentiation-related genes and quantitative RT-PCR of the MyoD gene in rQM7 and MyoD KO QM7#4 cells during differentiation. (D) Detection of myogenic differentiation-related genes in rQM7 and MyoD KO QM7#4 cells during differentiation. RT-PCR, reverse transcription-polymerase chain reaction. The images were observed under the inverted fluorescent microscope after staining with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI), scale bars = 200 μm.

Figure 5

String analysis of differentially expressed genes (DEGs) processed by RNA-sequencing data. (A) String analysis of total DEGs of rQM7 and MyoD KO QM7#4 cells during differentiation. (B) String analysis of DEGs significantly decreased in MyoD KO QM7#4 cells during differentiation. The red box indicates the MyoD transcript.