Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

Abstract

The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27–33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.

CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass

“…In this study, the ADG in mutant goats was 36% higher than that in WT goats (145 vs. 107 g) from D0 to D360, indicating that the growth rate of mutant goats was much higher than that of control goats, which is consistent with the ADG results of MSTN ‐edited sheep in our recent study (Wang et al . ). The BW of gene‐modified goats is significantly higher than that of several goat breeds in early growth stages, including at birth or weaning (Mahgoub & Lodge ; Mohammed & Amin ).…”

Biallelic β‐carotene oxygenase 2 knockout results in yellow fat in sheep via CRISPR/Cas9

“…By employing the CRISPR/Cas9 system, we successfully generated gene‐modified Tan sheep (Wang et al . ). Cas9 mRNA and two sgRNAs targeting exon 2 of the BCO2 gene were microinjected intracytoplasmatically into one cell stage sheep zygotes (Fig.…”

“…a, Table S1), which were collected surgically from naturally mated sheep as previously described by Wang et al . (). We were able to obtain a total of 10 Tan sheep with the BCO2 gene disrupted.…”

“…According to the previous sequencing results based on TA clones using whole blood samples (Wang et al . ), we defined founder animals without any wild type (WT) genotype as biallelic modified animals and founder animals with a monoallelic WT genotype as monoallelic modified animals. We next collected adipose tissues from the tails of 12‐month‐old sheep with different genotypes, which were subjected to T7E1 cleavage and Sanger sequencing.…”

CRISPR in livestock: From editing to printing

“…The knockout lambs were 25% heavier [7] and maintained the same wool quality traits than Merino lambs (unpublished data). Although this proof of concept did not include the study of the offspring, in following studies germline transmission of MSTN mutation have been reported by its presence in the gonads of founders (in sheep [46] and goats [47]) and offspring [47]. Amazingly, these early studies suggested that what farmers have pursued for centuries (i.e., animals producing both high-quality wool and more meat), might be achieved by CRISPR in few months.…”