College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Poult Sci. 2023 Oct;102(10):102970. doi: 10.1016/j.psj.2023.102970. Epub 2023 Jul 25.
The editing efficiency primarily hinders the utility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology in poultry. For a better understanding of the factors that influence the efficiency of gene knockout mediated by CRISPR/Cas9 in chicken DF1 cells, the single or dual single guide RNA (sgRNA) targeted exon regions of genes (taking anti-Müllerian hormone, TGF-beta receptor type-2 and Peroxisome proliferator-activated receptor gamma as examples) were designed. The sgRNA-CRISPR/Cas9 vectors with corresponding reporter vectors were transfected into DF1 cells. T7 endonuclease 1 (T7E1) and amplicon sequencing assay were compared for evaluating genome editing efficiency and the indel profiles were analyzed based on the data of amplicon sequencing. Meanwhile, to evaluate the precision of Cas9 cleavage, we also analyzed the homology of small insertion with the nucleotides of upstream and downstream of cleave sties. The surrogate reporter systems showed strong enrichment function, and the indel percentages were increased after puromycin selection. The indel ratios of T7E1 assay were lower than amplicon sequencing assay, which indicated T7E1 isn't fit to be used as the sole evaluation criterion for the targeting efficiency of CRISPR/Cas9. Based on the amplicon sequencing analysis, the editing efficiency showed noticeable differences among cells treated with different sgRNAs. However, the variety of indel efficiencies was not related to the GC content of sgRNA or chromosome types of targeted genes. The results showed that the dual sgRNA might not raise the indel ratios compared with individual sgRNA, but they could increase the ratios of the fragment deletions. The present study suggested that the surrogate reporter was an effective method to promote the editing efficiencies of CRISPR/Cas9 in chicken cells. The dual sgRNA could increase the fragment deletions, and the sensitivity of amplicon sequencing to detect cleavage was higher than the T7 endonuclease 1 assay. These results are essential to improve the application of CRISPR/Cas9 technology in chicken cells.
编辑效率主要阻碍了 clustered regularly interspaced short palindromic repeats (CRISPR) 技术在禽类中的应用。为了更好地理解影响 CRISPR/Cas9 介导的基因敲除在鸡 DF1 细胞中的效率的因素，设计了靶向基因外显子区域的单或双单向导 RNA（sgRNA）（以抗缪勒管激素、TGF-β受体型 2 和过氧化物酶体增殖物激活受体 γ为例）。将 sgRNA-CRISPR/Cas9 载体与相应的报告载体转染到 DF1 细胞中。比较 T7 内切酶 1（T7E1）和扩增子测序分析评估基因组编辑效率，并基于扩增子测序数据分析插入缺失图谱。同时，为了评估 Cas9 切割的精度，我们还分析了小插入与切割位点上下游核苷酸的同源性。替代报告系统显示出很强的富集功能，经嘌呤霉素选择后插入缺失百分比增加。T7E1 测定的插入缺失率低于扩增子测序分析，这表明 T7E1 不适合作为 CRISPR/Cas9 靶向效率的唯一评估标准。基于扩增子测序分析，不同 sgRNA 处理的细胞之间的编辑效率存在显著差异。然而，插入缺失效率的变化与 sgRNA 的 GC 含量或靶基因的染色体类型无关。结果表明，与单个 sgRNA 相比，双 sgRNA 不一定能提高插入缺失率，但能增加片段缺失的比例。本研究表明，替代报告是提高 CRISPR/Cas9 在鸡细胞中编辑效率的有效方法。双 sgRNA 可以增加片段缺失，而扩增子测序对切割的检测灵敏度高于 T7 内切酶 1 测定。这些结果对提高 CRISPR/Cas9 技术在鸡细胞中的应用至关重要。
