Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, Munich, Germany.
Graduate School of Systemic Neurosciences, LMU Munich, Planegg-Martinsried, Germany.
Nat Protoc. 2021 Mar;16(3):1714-1739. doi: 10.1038/s41596-020-00481-2. Epub 2021 Feb 17.
The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 d.
最近的 CRISPR 革命为研究人员提供了强大的工具，可在多种生物体中进行基因组编辑。然而，最近的报告表明，在编辑后的小鼠和人类诱导多能干细胞 (iPSC) 中，广泛存在标准质量控制无法检测到的非预期 CRISPR 诱导的靶标效应 (OnTE)。通过改变靶向或邻近基因的表达，OnTE 会严重影响 CRISPR 编辑细胞和生物体的表型，从而导致数据解读错误，这可能会破坏基于 CRISPR 的研究的可靠性。在这里，我们描述了一种广泛适用于检测非同源末端连接介导和同源定向修复介导编辑后基因组编辑细胞和生物体中的 OnTE 的框架。我们的方案可识别 OnTE，如大片段缺失、大片段插入、重排或杂合性丢失 (LOH)。这是通过首先对基因组 DNA 进行定量基因分型 PCR (qgPCR) 来实现的，该方法使用已针对基因分型进行优化的相同 PCR 扩增子来确定靶位点的完整等位基因数量。这种基因分型和定量的组合使得能够排除单等位基因 OnTE 和半合子编辑的克隆，这些克隆在标准 Sanger 测序中通常被错误地描述为正确编辑。其次，通过对编辑位点附近的单核苷酸多态性 (SNP) 进行基因分型，检测 LOH 的发生，这可以使用基于 Sanger 测序的方法或 SNP 微阵列来实现。所有步骤都经过优化，以最大限度地提高简单性和降低成本，从而促进该方案在整个领域的广泛传播和适用性。从基因组 DNA 提取到排除 OnTE 的整个过程可以在 6-9 天内完成。
