NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW, 2568, Australia.
Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, Camden, NSW, Australia.
BMC Res Notes. 2024 Jul 26;17(1):205. doi: 10.1186/s13104-024-06861-1.
Insertion and deletion (indel) analysis of CRISPR-Cas guide RNAs (gRNAs) is crucial in gene editing to assess gRNA efficiency and indel frequency. This study evaluates the utility of CRISPResso2 with Oxford Nanopore sequencing data (nCRISPResso2) for gRNA indel screening, compared to two common Sanger sequencing-based methods, TIDE and ICE. To achieve this, sheep and horse fibroblasts were transfected with Cas9 and a gRNA targeting the myostatin (MSTN) gene. DNA was subsequently extracted, and PCR products exceeding 600 bp were sequenced using both Sanger and Nanopore sequencing. Indel profiling was then conducted using TIDE, ICE, and nCRISPResso2.
Comparison revealed close correspondence in indel formation among methods. For the sheep MSTN gRNA, indel percentages were 52%, 58%, and 64% for TIDE, ICE, and nCRISPResso2, respectively. Horse MSTN gRNA showed 81%, 87%, and 86% edited amplicons for TIDE, ICE, and nCRISPResso2. The frequency of each type of indel was also comparable among the three methods, with nCRISPResso2 and ICE aligning the closest. nCRISPResso2 offers a viable alternative for CRISPR-Cas gRNA indel screening, especially with large amplicons unsuitable for Illumina sequencing. CRISPResso2's compatibility with Nanopore data enables cost-effective and efficient indel profiling, yielding results comparable to common Sanger sequencing-based methods.
在基因编辑中，CRISPR-Cas 引导 RNA（gRNA）的插入和缺失（indel）分析对于评估 gRNA 效率和 indel 频率至关重要。本研究评估了 CRISPResso2 与牛津纳米孔测序数据（nCRISPResso2）在 gRNA indel 筛选中的应用，与两种常用的基于 Sanger 测序的方法 TIDE 和 ICE 进行了比较。为了实现这一目标，绵羊和马成纤维细胞被转染 Cas9 和针对肌肉生长抑制素（MSTN）基因的 gRNA。随后提取 DNA，并使用 Sanger 和纳米孔测序对超过 600 bp 的 PCR 产物进行测序。然后使用 TIDE、ICE 和 nCRISPResso2 进行 indel 分析。
比较结果显示，不同方法在 indel 形成方面具有高度一致性。对于绵羊 MSTN gRNA，TIDE、ICE 和 nCRISPResso2 的 indel 百分比分别为 52%、58%和 64%。马 MSTN gRNA 的编辑扩增子在 TIDE、ICE 和 nCRISPResso2 中的 indel 百分比分别为 81%、87%和 86%。三种方法中每种类型的 indel 频率也相似，nCRISPResso2 和 ICE 最为接近。nCRISPResso2 是一种可行的 CRISPR-Cas gRNA indel 筛选替代方法，特别是对于不适合 Illumina 测序的大扩增子。nCRISPResso2 与纳米孔数据的兼容性使得成本效益高且高效的 indel 分析成为可能，其结果与常见的基于 Sanger 测序的方法相当。
