Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs
Authors: Xuan Shi
Linfang Yang
Wu Zeng
Min Wang
Xiaohong Liu
Delin Mo
Ruiqiang Li
Hongbo Liu
Yaosheng Chen
Xiaofeng Liu
Miao Ma
Zixuan Wei
Jianhua Zeng
Zuyong He
Journal: Transgenic Research (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: CRISPR/Cas9; Liang Guang Small Spotted pig; Muscle mass; Myostatin; Signal peptide.
Affiliations: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, No. 3 Road of Higher Education Mega Centre North, Guangzhou, 510006, People's Republic of China., Guangdong YIHAO Food Co., Ltd., Guangzhou, 510620, People's Republic of China., State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, No. 3 Road of Higher Education Mega Centre North, Guangzhou, 510006, People's Republic of China. .

Abstract:  Myostatin (MSTN), a member of the transforming growth factor-β superfamily, is a negative regulator of muscle growth and development. Disruption of the MSTN gene in various mammalian species markedly promotes muscle growth. Previous studies have mainly focused on the disruption of the MSTN peptide coding region in pigs but not on the modification of the signal peptide region. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was used to successfully introduce two mutations (PVD20H and GP19del) in the MSTN signal peptide region of the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Both mutations in signal peptide increased the muscle mass without inhibiting the production of mature MSTN peptide in the cells. Histological analysis revealed that the enhanced muscle mass in MSTN+/PVD20H pig was mainly due to an increase in the number of muscle fibers. The expression of MSTN in the longissimus dorsi muscle of MSTN+/PVD20H and MSTNKO/PVD20H pigs was significantly downregulated, whereas that of myogenic regulatory factors, including MyoD, Myogenin, and Myf-5, was significantly upregulated when compared to those in the longissimus dorsi muscle of wild-type pigs. Meanwhile, the mutations also activated the PI3K/Akt pathway. The results of this study indicated that precise editing of the MSTN signal peptide can enhance porcine muscle development without markedly affecting the expression of mature MSTN peptide, which could exert other beneficial biological functions in the edited pigs. 

Retracted: No

PubPeer comments:
 No off-target analysis done. Just saying  precise [does not make it] so - no proper sequencing done, and whatever was done has not been submitted. One can do something as simple as [amplicon sequencing] - since they are doing so many other things. , 
 Here, the precise means the in frame editing of the signal peptide of MSTN, other than random indels generated by CRISPR Cas9 in this gene. The  precise here does not refer to no off-target editing in the genome of edited pigs, as we have not yet carried out the genome-wide off-target editing analysis. The traditional used amplicon sequencing actually has limited capability in detection of potentials off-target editing, thus we did not use this method for off-target analysis in this study. , 
 #1 I m sorry for misunderstanding  amplicon sequencing as Sanger sequencing of cloned PCR products. Amplicon sequencing is useful for analysis of genetic variation in specific genomic regions (e.g. characterization of editing results in target site by CRISPR Cas9), but not suitable for genow-wide screening of potential off-targets. , 
 1. Yes, amplicon-seq is not sufficient for finding genome wide off-targets - but it is better than nothing (to show precision of the edits - and sometimes [even off-targets] ). 2. Even [WGS is not sufficient] to find off-targets - if not done deep enough. 3. Precise usually means  nothing else went wrong - we dont really know what happened in the rest of the genome, do we? , 
 #1 On your blog you say  20 21 deaths in a Chinese CRISPR PD-1 k o trial. Our cells just cant take so much genomic damage. Saying these terminal cancer patients died of CRISPR-induced DNA damage does not make it so. Terminal cancer patients most likely died of cancer, not side effects of CRISPR. ,