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Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System
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Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality.

Shiwei Zhou1,2, Peter Kalds1,3, Qi Luo1

  • 1Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, China.

BMC Genomics
|May 6, 2022
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Summary

This study optimized CRISPR/Cas9 gene editing to create homozygous myostatin (MSTN) knockout sheep, improving growth without affecting meat quality. This advances livestock genetic engineering for enhanced agricultural productivity.

Keywords:
CRISPR/Cas9 optimizationGenome editingHomozygous gene knockoutMSTNMuscle growthSheep

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Area of Science:

  • Livestock genetics
  • Genome editing technologies
  • Animal breeding

Background:

  • CRISPR/Cas9 systems enable precise genetic modification in livestock for agriculture and biomedicine.
  • Previous methods often resulted in heterozygous gene edits, limiting desired phenotypic outcomes.
  • Efficient generation of homozygous gene-edited animals is crucial for agricultural applications.

Purpose of the Study:

  • To optimize CRISPR/Cas9 delivery for efficient generation of homozygous myostatin (MSTN) knockout sheep.
  • To evaluate the impact of MSTN gene editing on sheep growth and meat quality.

Main Methods:

  • Utilized sgRNA selection software (sgRNAcas9) to identify effective sgRNAs targeting the MSTN gene.
  • Validated ten sgRNAs in vitro using sheep fibroblast cells, selecting four with >50% efficiency.
  • Determined optimal Cas9 mRNA and sgRNA concentrations (100 ng/μL and 200 ng/μL, respectively) for microinjection into sheep embryos.

Main Results:

  • Achieved approximately 50% targeting efficiency and 25% homozygous knockout efficiency for the MSTN gene in sheep.
  • MSTN-knockout lambs demonstrated increased body weight and average daily gain compared to wild-type controls.
  • Key meat quality parameters (pH, drip loss, intramuscular fat, crude protein, shear force) remained unchanged in MSTN-knockout lambs.

Conclusions:

  • In vitro evaluation of sgRNAs and microinjection dosage is critical for optimizing gene editing efficiency.
  • This optimized approach successfully generated homozygous knockout sheep.
  • MSTN gene editing enhances livestock productivity without compromising meat quality, supporting its use in farm animal improvement.