Abstract
Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.
Highlights
The high efficiencies strictly apply only to the introduction of non-defined, random mutations as a result of non-homologous end joining repair (NHEJ)
Several recent studies have reported the generation of marker-free livestock by either microinjection of mRNA or protein of nucleases or somatic cell nuclear transfer using mutant cell clones free of SMGs
Some GM livestock produced with the introduction of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEn) or CRISPR/Cas[9] into zygote or primary cells were found to be mosaic and contain multiple different mutations with many of these mutations not resulting in the intended functional disruption of the target genes[2,3,4,23,24,25]
Summary
The high efficiencies strictly apply only to the introduction of non-defined, random mutations as a result of non-homologous end joining repair (NHEJ). Homozygous MSTN mutant mice were up to 30% larger in body weight and their muscles had larger cross-sectional fiber areas (hypertrophy) as well as a greater number of fibers (hyperplasia) compared to wild-type controls[15]. All these observations suggest that MSTN is an ideal target for increasing muscle growth and improving meat production by genetic manipulation in livestock. Molecular tests proved the functional disruption of MSTN and the removal of the SMG in the cloned founders These isozygous, SMG-free MSTN KO pigs exhibited elevated level of myogenic regulatory factors, muscle hyperplasia, overgrown skeleton muscle and decreased fat mass, which duplicated the “double muscling” phenotype. This work provides the basis for the commercial production of GM elite pigs with increased consumers’ confidence of the absence of biological risks
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