Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (RNA-Seq). Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (RNA-Seq)

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering. Overall design: Nanopore sequencing analysis of high molecular weight genomic DNA surrounding CRISPR RNA (crRNA) target sites, using Cas9-based enrichment of targeted regions.

多重遗传扰动是探究编码或非编码遗传元件之间功能互作的关键手段。相较于双链DNA切割技术，基于CRISPR干扰 (CRISPR interference, CRISPRi) 的染色质阻抑形成策略可避免遗传毒性，且在混池筛选实验中对非编码调控元件的扰动效果更优。然而，当前的CRISPRi混池筛选方法仅能实现单个细胞靶向1~3个基因组位点。本研究构建了一种氨基酸球菌属Cas12a (AsCas12a) 突变体——多重转录干扰型AsCas12a (multiAsCas12a)，该突变体携带R1226A突变，该突变可通过DNA切口化稳定核糖核蛋白-DNA复合物。相较于核酸酶失活型AsCas12a-KRAB融合蛋白，multiAsCas12a-KRAB融合蛋白可提升CRISPRi活性，通常能挽救那些与后者联用时无活性的慢病毒递送型CRISPR RNA (crRNA) 的活性。multiAsCas12a-KRAB可在高通量混池筛选中支持基于6重crRNA阵列的CRISPRi操作。借助multiAsCas12a-KRAB，我们在人类细胞中鉴定出增强子元件，并解析了顺式调控元件的组合功能。本研究结果构建了一套群体测试框架，可高效筛查大量染色质扰动组合，用于生物学发现与工程化改造。整体实验设计：基于Cas9靶向区域富集技术，对CRISPR RNA (crRNA) 靶位点周边的高分子量基因组DNA进行纳米孔测序 (Nanopore sequencing) 分析。