Myostatin site‐directed mutation and simultaneous PPARγ site‐directed knockin in bovine genome

Abstract

Most studies on the acquisition of advantageous traits in transgenic animals only focus on monogenic traits. In practical applications, transgenic animals need to possess multiple advantages. Therefore, multiple genes need to be edited simultaneously. CRISPR/Cas9 technology has been widely used in many research fields. However, few studies on endogenous gene mutation and simultaneous exogenous gene insertion performed via CRISPR/Cas9 technology are available. In this study, the CRISPR/Cas9 technology was used to achieve myostatin (MSTN) point mutation and simultaneous peroxisome proliferator‐activated receptor‐γ (PPARγ) site‐directed knockin in the bovine genome. The feasibility of this gene editing strategy was verified on a myoblast model. The same gene editing strategy was used to construct a mutant myoblast model with MSTN mutation and simultaneous PPARγ knockin. Quantitative reverse‐transcription polymerase chain reaction, immunofluorescence staining, and western blot analyses were used to detect the expression levels of MSTN and PPARγ in the mutant myoblast. Results showed that this strategy can inhibit the expression of MSTN and promote the expression of PPARγ. The cell counting kit‐8 cell proliferation analysis, 5‐ethynyl‐2′‐deoxyuridine cell proliferation analysis, myotube fusion index statistics, oil red O staining, and triglyceride content detection revealed that the proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities of the mutant myoblasts were higher than those of the wild myoblasts. Finally, transgenic bovine embryos were obtained via somatic cell nuclear transfer. This study provides a breeding material and technical strategy to breed high‐quality bovine and a gene editing method to realize the mutation of endogenous genes and simultaneous insertion of exogenous genes in genomes.

Genetic Variations and Haplotypic Diversity in the Myostatin Gene of Different Cattle Breeds in Russia

“…Currently, searches are being carried out to identify a method of obtaining animals with knock-out myostatin using CRISPR/CAS9 technology [32]. However, in reality, the selection of animals based on genotyping information seems to be the most affordable way to increase the productivity of beef breeds of cattle.…”

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

“…At two years old, the body weights of MSTN -edited and WT individuals were 593.59 and 485.73 kg, respectively (10.2% and 16.8% increase rates at 12 and 24 months, respectively) . Of these CRISPR/Cas9 studies, an article reported the attempt to generate simultaneous MSTN point mutation and PPARγ knockin at the embryonic level to increase muscle mass through MSTN editing and potentially archive improved intramuscular fat deposition through the PPARγ overexpression . Additionally, stable germline transmission of MSTN -edited cattle was reported, and an attempt to induce additional gene ( PRNP ) editing to MSTN -edited germ cells using electroporation was achieved .…”

“…35 Of these CRISPR/ Cas9 studies, an article reported the attempt to generate simultaneous MSTN point mutation and PPARγ knockin at the embryonic level to increase muscle mass through MSTN editing and potentially archive improved intramuscular fat deposition through the PPARγ overexpression. 32 Additionally, stable germline transmission of MSTN-edited cattle was reported, and an attempt to induce additional gene (PRNP) editing to MSTN-edited germ cells using electroporation was achieved. 36 This could likely contribute to the generation of improved muscle mass by targeting the MSTN gene and potential resistance to prion infection by editing the PRNP gene.…”

“…TALENs were also applied to target the bovine MSTN gene, − and live TALENs-mediated MSTN knockout cattle were generated using ovum pickup, in vitro fertilization, and zygote microinjection (OPU-IVF-ZM) (Table ). More recently, CRISPR/Cas9 was applied to edit the bovine MSTN gene in vitro − and in vivo − (Table ). The generated CRISPR/Cas9-induced MSTN -targeted cattle exhibited the expected increased muscle mass phenotype.…”

Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals

“…MSTN, on the other hand, is a muscle development negative regulator that inhibits muscle cell multiplication and differentiation, as well as muscle development and formation. As a result, this research on using CRISPR/Cas9 to achieve myostatin point mutation and PPAR site guided knock-in within the bovine genome is remarkable [ 132 ].…”