The Jackson Laboratory, Bar Harbor, Maine, ME, USA.
The Centre for Computational Medicine, The Hospital for Sick Children, Toronto, ON, Canada.
Commun Biol. 2023 Jun 10;6(1):626. doi: 10.1038/s42003-023-04974-0.
Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.
使用 CRISPR 相关（Cas）蛋白进行基因组编辑为“纠正”导致遗传疾病的变体提供了巨大的希望。为了实现这一承诺，在编辑过程中不能发生脱靶基因组变化。在这里，我们使用全基因组测序将 50 只 Cas9 编辑的创始小鼠的基因组与 28 只未处理的对照小鼠的基因组进行比较，以评估酿脓链球菌 Cas9 诱导的脱靶诱变的发生。全基因组测序数据的计算分析在 23 个预测的脱靶位点检测到 18/163 个指南的 26 个独特序列变体。虽然计算检测到的变体在 15/50 个 Cas9 基因编辑创始动物中被识别，但只有 8/15 个创始动物中的 38%（10/26）的变体通过 Sanger 测序验证。Cas9 脱靶活性的体外测定仅鉴定出基因组测序数据中存在的两个未预测的脱靶位点。总的来说，只有 4.9%（8/163）的指南具有可检测的脱靶活性，每个分析的创始动物中有 0.2 个 Cas9 脱靶突变。相比之下，无论 Cas9 是否暴露于基因组，我们在每只小鼠中都观察到约 1100 个独特变体，表明脱靶变体在 Cas9 编辑小鼠的遗传异质性中仅占很小的一部分。这些发现将为未来 Cas9 编辑动物模型的设计和使用提供信息，并为评估遗传多样化患者群体中的脱靶潜力提供背景。
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