Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

Abstract

Background CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs. Conclusions This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.

When Less Is More: Targeting the Myostatin Gene in Livestock for Augmenting Meat Production

“…In our optimized system, it was shown that microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency, and MSTN -knockout sheep were generated with approximately 50% targeting efficiency and a homozygous knockout efficiency of 25% . The average birth weight of MSTN -knockout lambs was higher than that of WT lambs, and MSTN knockout lambs exhibited remarkably higher body weight in subsequent developmental stages . Moreover, main meat quality characteristics, including pH, drip loss, intramuscular fat, crude protein, and shearing force of gluteal muscles, did not show changes between the MSTN -edited and WT lambs .…”

“…In our laboratory, we have targeted the ovine MSTN gene several times . These include multiplex MSTN editing with the ASIP and BCO2 genes, induction of large genomic deletions within MSTN , and optimization of the Cas9:sgRNA delivery to generate bi-allelic MSTN knockout sheep . In our optimized system, it was shown that microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency, and MSTN -knockout sheep were generated with approximately 50% targeting efficiency and a homozygous knockout efficiency of 25% .…”

“…These include multiplex MSTN editing with the ASIP and BCO2 genes, induction of large genomic deletions within MSTN , and optimization of the Cas9:sgRNA delivery to generate bi-allelic MSTN knockout sheep . In our optimized system, it was shown that microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency, and MSTN -knockout sheep were generated with approximately 50% targeting efficiency and a homozygous knockout efficiency of 25% . The average birth weight of MSTN -knockout lambs was higher than that of WT lambs, and MSTN knockout lambs exhibited remarkably higher body weight in subsequent developmental stages .…”

“…( l ) CRISPR/Cas9-mediated MSTN -edited Merino sheep [WT (up) and MSTN -edited (down)] . ( m ) CRISPR/Cas9-mediated MSTN -edited Chinese Tan sheep . ( n ) TALENs-mediated MSTN -edited goats .…”

Inactivation of the MSTN gene expression changes the composition and function of the gut microbiome in sheep

“…Inactivation of MSTN expression causes muscle hyperplasia and hypertrophy, which has been observed in mice [1], cattle [2][3][4], dogs [5,6], sheep [7], goats [8,9], pigs [10,11] and even humans [12]. In view of the great value of MSTN in animal breeding, we produced the MSTN-edited Tan sheep by CRISPR/Cas9, and found that editing of the MSTN significantly increased diameter of myofibers, the average daily gain (ADG) and body weight in sheep [13,14].…”