Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect

Abstract

ObjectiveBased on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells.MethodsChicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP) gene and targeted multiplex guide RNAs (gRNAs), the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure.ResultsThrough the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed.ConclusionThe knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

Effect of in Ovo Injection of MyoPro Gene on Growth Parameter in Fayoumi Chicken

“…Several studies were done by myostatin inhibitors in cattle. These showed muscle doubling (Kambadur et al, 1997, Rodgers &Garikipati, 2008 and showed muscular hyperplasia (Wegner et al, 2000) There is no previous studies using Myo pro in chicken so the present work considered the first trail in chicken However, Lee et al, (2017) using Cas9 (Amthor et al, 2002, Joulia et al, 2003, Hayashi et al, 2008and Muroya et al, 2009. Binding regions for muscle growth related transcription factors MyoD were identified in the promoter of myostatin gene (Salerno et al 2004).…”

CRISPR/Cas9 gene editing in a chicken model: current approaches and applications

“…In these studies, the myostatin (MSTN) gene was disrupted using targeted editing technology, which led to enhanced formation of skeletal muscles, and this could be beneficial for meat production. The first approach to knockout the MSTN gene in chicken DF-1 cells has been demonstrated by Lee et al (2017). The authors used the Cas9-D10A nickase of mutated CRISPR/Cas9 to efficiently modulate the MSTN gene.…”

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

“…CRISPR technologies have greatly influenced biological, biomedical, and agricultural investigations. This technology can efficiently create numerous genetic mutations, including gene KO and knock-in mutants, to generate genetically modified cells, perform postnatal gene editing, and generate germline transgenic animals [19][20][21]. At present, this technology is not widely employed in agricultural animals like chickens to improve traits and breeding.…”