Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus; Gates Institute, University of Colorado School of Medicine, Anschutz Medical Campus.
Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Anschutz Medical Campus.
J Vis Exp. 2024 Nov 1(213). doi: 10.3791/67069.
Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.
通过测序进行体外报告切割效果的环形化（CIRCLE-seq）是一种新开发的技术，用于通过靶向测序 CRISPR-Cas9 切割的 DNA 来公正地鉴定 CRISPR-Cas9 的非预期切割位点。该方案涉及环状化基因组 DNA（gDNA），随后用 Cas9 蛋白和感兴趣的指导 RNA（gRNA）处理。处理后，将切割的 DNA 纯化并制备为用于 Illumina 测序的文库。测序过程生成配对末端读数，提供每个切割位点的全面数据。CIRCLE-seq 相对于其他体外方法具有几个优势，包括最小的测序深度要求、低背景和 Cas9 切割 gDNA 的高富集度。这些优势提高了识别预期和非预期切割事件的灵敏度。本研究提供了一种使用 CIRCLE-seq 检查 CRISPR-Cas9 脱靶活性的全面、逐步的程序。例如，通过在 AAVS1 基因座修饰过程中绘制 CRISPR-Cas9 的全基因组非预期切割位点，验证了该方案。整个 CIRCLE-seq 过程可以在两周内完成，为细胞生长、DNA 纯化、文库制备和 Illumina 测序提供了足够的时间。将测序数据输入 CIRCLE-seq 管道有助于简化对切割位点的解释和分析。
