Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, Oregon, USA; Portland, Oregon, USA.
Department of Pediatrics, University of Washington, Seattle, Washington, USA; and Portland, Oregon, USA.
CRISPR J. 2022 Aug;5(4):548-557. doi: 10.1089/crispr.2021.0140. Epub 2022 Jul 12.
Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.
靶向测序仍然是临床和研究应用的一种有价值的技术。然而，许多现有的技术存在广泛的鸟嘌呤-胞嘧啶（GC）序列含量偏差、高输入 DNA 要求和定制面板的高成本等问题。我们开发了 Cas12a-Capture，这是一种低成本且高度可扩展的靶向测序方法。该方法利用预先编程的 guide RNA 引导 Cas12a 切割双链 DNA，然后利用产生的四到五个核苷酸突出端，与定制的测序接头进行选择性连接。添加第二个测序接头并富集连接产物，即可生成靶向序列文库。我们首先使用 7176 个靶向 3.5Mb DNA 的 guide 进行了试点实验。利用这些数据，我们对 Cas12a-Capture 效率的序列决定因素进行了建模，然后设计了一组优化的 11438 个靶向 3.0Mb 的 guide。优化后的 guide 集实现了靶向区域的平均 64 倍富集，最小化了 GC 偏差。Cas12a-Capture 的变体调用与 Illumina Platinum Genome 的调用具有很强的一致性，特别是对于单核苷酸变体，可以通过应用基本的变体质量启发式规则来改进。我们认为 Cas12a-Capture 具有广泛的潜在临床和研究应用，并可适用于任何双链 DNA 模板或基因组的选择性富集。
