Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%–99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

标准 CRISPR 介导的基因破坏策略依赖于 Cas9 诱导的 DNA 双链断裂 (DSB)。在这里，我们展示了 CRISPR 依赖性碱基编辑通过精确地将四个密码子（CAA、CAG、CGA 和 TGG）转换为终止密码子而不形成 DSB 来有效地灭活基因。为了通过诱导终止密码子 (iSTOP) 促进基因失活，我们提供了超过 340 万个单引导 RNA (sgRNA) 的数据库，iSTOP (sgSTOPs) 靶向八个真核物种中 97%–99% 的基因，并且我们描述限制性片段长度多态性 (RFLP) 检测，可快速检测细胞群和克隆中 iSTOP 介导的编辑。为了简化 sgSTOP 的选择，我们的资源包括脱靶倾向的注释、目标亚型的百分比、无义介导的衰变的预测以及用于 RFLP 分析的限制性内切酶。此外，我们的数据库包括 sgSTOP，可用于精确建模超过 32,000 个与癌症相关的无义突变。总而言之，这项工作为 iSTOP 的无 DSB 基因破坏提供了全面的资源。