title: Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect.
abstract: OBJECTIVE
  Based 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.
  
  
  METHODS
  Chicken 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.
  
  
  RESULTS
  Through 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
authors.forename: Jeong Hyo
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
authors.forename: Si Won
authors.initials: SW
authors.lastname: Kim
authors.affiliation: Graduate School of International Agricultural Technology and Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchang 25354, Korea.
authors.forename: Tae Sub
authors.initials: TS
authors.lastname: Park
keywords: Cas9; Genome Editing; Knockout; Myostatin; Nickase; Poultry