Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, China.
College of Veterinary Medicine, Northwest A&F University, Yangling, China.
BMC Genomics. 2022 May 6;23(1):348. doi: 10.1186/s12864-022-08594-6.
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.
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.
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.
基于 CRISPR/Cas9 的基因组编辑系统已被用于高效地对家畜进行基因改造，以实现生物医学和农业应用的精确遗传改变。此前，我们通过单细胞胚胎显微注射 Cas9 mRNA 和单指导 RNA（sgRNA）混合物成功地生成了基因编辑绵羊和山羊。然而，使用这种方法产生的大多数基因编辑动物都是杂合子。此外，非纯合的基因编辑结果可能无法以有效的方式完全产生所需的表型。
我们报告了优化 Cas9 mRNA-sgRNA 递送系统的方法，以高效生成肌肉生长抑制素（MSTN）基因敲除绵羊，从而提高生长速度和肉产量。首先，使用 sgRNA 选择软件（sgRNAcas9）对 sgRNA 进行了初步筛选，以获得高效 sgRNA。选择了 10 个针对 MSTN 基因的 sgRNA，并在绵羊成纤维细胞中进行了体外验证。10 个 sgRNA 中有 4 个（2 个在 exon1，2 个在 exon2）的靶向效率>50%。为了确定最佳的 CRISPR/Cas9 显微注射浓度，我们将 Cas9 mRNA 和 sgRNA 混合物的四个浓度水平和三个水平分别注射到绵羊胚胎中。显微注射 100ng/μL Cas9 mRNA 和 200ng/μL sgRNAs 可获得最高的靶向效率。此外，使用高效 sgRNA 和最佳显微注射浓度，以大约 50%的靶向效率生成 MSTN 基因敲除绵羊，达到 25%的纯合敲除效率。还研究了 MSTN 编辑羔羊的生长速度和肉质。MSTN 基因敲除羔羊的体重和平均日增重增加。此外，与野生型羔羊相比，MSTN 基因敲除羔羊的臀肌 pH 值、滴水损失、肌内脂肪、粗蛋白和剪切力没有变化。
本研究强调了体外评估对 sgRNA 优化和基因编辑试剂显微注射剂量的重要性。这种方法使纯合基因敲除绵羊的高效工程成为可能。此外，本研究证实 MSTN 基因敲除羔羊不会对肉质产生负面影响，因此支持将基因编辑作为提高农场动物生产力的工具。
