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.
Highlights
Since transgenic mice were successfully created by introducing a foreign gene construct [1], genetic modification systems to generate transgenic, knockout and knockin animals have been the most promising approaches that help advance a comprehensive understanding of biology and lead to practical applications in agriculture and biopharmacy
clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)-D10A nickase-mediated myostatin knockout in chicken DF1 cells Based on the genome sequence information [NCBI, Gallus gallus MSTN, NM_001001461 and UCSC Genomics Institute, chicken BLAT], the target guide RNA sites were designed following protospacer adjacent motif (PAM) sequences (Figure 1)
Based on the somatic cell nuclear transfer (SCNT) cloning or direct injection technique into one-cell-stage embryos, CRISPR/Cas9-mediated knockout and knockin individuals have been successfully generated in various mammals [13,15,16]
Summary
Since transgenic mice were successfully created by introducing a foreign gene construct [1], genetic modification systems to generate transgenic, knockout and knockin animals have been the most promising approaches that help advance a comprehensive understanding of biology and lead to practical applications in agriculture and biopharmacy. The recently developed transgenic technique utilizing piggyBac transposon and transposase is the most promising protocol to produce transgenic chickens with a stable transgene expression without tissue-specific repression as well as an efficient transgene insertion into genomic structure [2,3]. Since knockout mice have been generated by conventional homologous recombination [5,6,7,8], the knockout system has revolutionized the research field of functional genomics by allowing the analysis of specific gene function(s) in animals.
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