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在多种生物体中进行基因组编辑的强大工具。然而，最近的报告表明，在小鼠和人类诱导多能干细胞 (iPSC) 的编辑位点普遍存在 CRISPR 诱导的非预期靶向效应 (OnTE)，这些效应逃脱了标准质量控制。通过改变目标基因或邻近基因的基因表达，OnTE 可以严重影响 CRISPR 编辑的细胞和生物体的表型，从而导致数据误解，从而破坏基于 CRISPR 的研究的可靠性。在这里，我们描述了一个广泛适用的框架，用于在非同源末端连接介导和同源定向修复介导的编辑后检测基因组编辑细胞和生物体中的 OnTE。我们的协议能够识别 OnTE，例如大删除、大插入、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 PCR 使用已针对基因分型进行优化的相同 PCR 扩增子来确定目标位点完整等位基因的数量。这种基因分型和定量的结合使得排除具有单等位基因 OnTE 和半合子编辑的克隆成为可能，这些克隆通常在标准桑格测序中被错误地描述为正确编辑。其次，使用基于桑格测序的方法或 SNP 微阵列，通过对邻近的单核苷酸多态性 (SNP) 进行基因分型来检测编辑基因座周围是否存在 LOH。所有步骤都经过优化，以最大限度地简化并最大限度地降低成本，以促进在整个领域的广泛传播和适用性。从基因组 DNA 提取到 OnTE 排除的整个方案可在 6-9 天内完成。