Background One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

中文翻译：

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免扩增长读长测序揭示了不可预见的 CRISPR-Cas9 脱靶活性


背景 对 CRISPR-Cas9 基因组编辑的一个持续关注是非特异性引导 RNA (gRNA) 结合可能会诱导脱靶突变。然而，准确预测 CRISPR-Cas9 脱靶活性具有挑战性。在这里，我们提出了 SMRT-OTS 和 Nano-OTS，这是两种新颖的、无扩增、长读长的测序方案，用于在体外检测 Cas9 对 gRNA 驱动的基因组 DNA 的消化。结果 该方法使用人类细胞系 HEK293 进行评估，并使用高度准确的 HiFi SMRT 读数以 18 倍覆盖度重新测序。 SMRT-OTS 和 Nano-OTS 首先应用于靶向 HEK293 基因组 DNA 的三种不同 gRNA，从而通过两种方法鉴定出一组 55 个高置信度 gRNA 切割位点。其中 25 个位点没有被脱靶预测软件报告，因为与人类参考相比，它们包含四个或更多单核苷酸错配或插入/删除错配。其他实验表明，85% 的 Cas9 切割位点也可以通过其他体外方法找到，并且在短读长无法唯一比对的基因体中可以检测到靶标和脱靶位点。尽管 SMRT-OTS 和 Nano-OTS 识别了多个先前在细胞中经过验证的脱靶编辑活性的位点，但我们自己在人类成纤维细胞中进行的 CRISPR-Cas9 编辑实验并未在体外预测的位点产生可检测到的脱靶突变。 。然而，插入缺失和结构变异事件在目标位点处丰富。结论 免扩增长读长测序揭示了使用计算工具难以预测的体外 Cas9 切割位点，包括短读长测序无法访问的暗基因组区域。