Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

Abstract

Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

Maternal RNF114-mediated target substrate degradation regulates zygotic genome activation in mouse embryos

“…The destruction of the RING domain in RNF114 may result in its degradation because the key domains are important to protein stability (Heessen et al, 2005;Liu et al, 2017). The instability of mutant mRNA was unexpected, although the degradation of mutant mRNA had been previously demonstrated in some gene-edited animals (Oo et al, 2020;Xu et al, 2020). The female mice were completely sterile after Rnf114 editing.…”

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens

“…Studies to date have examined the gain and loss‐of‐function of MSTN in chicken embryos, the consequences for the muscle progenitor population, and the association between MSTN SNPs and broiler performance 39,40 . We did not further analyze the our MSTN genome‐modified chickens in detail, but recent studies showed that mutation of MSTN in chicken 41 and quail has an anti‐myogenic effect 42 ; moreover, spatial MSTN mutations introduced by adenoviral CRISPR‐Cas9 induce differential gene expression involved in cell differentiation, muscle growth and development, and energy metabolism in postnatal chicken 43 . Therefore, we expect that these animals will exhibit elevated protein synthesis capacity as well as improved muscle cell development.…”

Precise Genome Editing in Poultry and Its Application to Industries

“…These results are important because the germline chimera efficiency is higher than those of other PGC-free methods, and the method has the potential to be broadly applied across poultry species. Additionally, adenovirus can be applied to postnatal gene editing in poultry [ 67 ]. Xu et al adenoviral vector expressing MSTN gene-targeting sgRNA and SpCas9 was injected into the chick leg muscle, resulting in successful knockout of MSTN in that tissue.…”