Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (Nanopore)

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. Nanopore sequencing analysis of high molecular weight genomic DNA surrounding CRISPR RNA (crRNA) target sites, using Cas9-based enrichment of targeted regions.

多重遗传扰动是检测编码与非编码遗传元件间功能互作的核心手段。相较于双链DNA切割技术，基于CRISPR干扰（CRISPR interference, CRISPRi）的染色质阻遏形成策略可规避遗传毒性，且在混合筛选实验中更适于扰动非编码调控元件。然而，当前CRISPRi混合筛选方法仅能实现每个细胞靶向1~3个基因组位点。本研究开发了一种氨基酸球菌属Cas12a（Acidaminococcus Cas12a, AsCas12a）变体——多重转录干扰型AsCas12a（multiplexed transcriptional interference AsCas12a, multiAsCas12a），其引入R1226A突变，该突变可通过DNA切口形成稳定的核糖核蛋白-DNA复合物。相较于核酸酶失活的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失活的慢病毒递送CRISPR RNA（crRNA）的活性。multiAsCas12a-KRAB可在高通量混合筛选中借助6重crRNA阵列实现CRISPRi应用。本研究利用multiAsCas12a-KRAB在人类细胞中发现了增强子元件，并解析了顺式调控元件的组合功能。上述成果构建了一套分组检测框架，可高效筛查大量染色质扰动组合以用于生物学发现与工程化改造。本研究还采用基于Cas9的靶向富集技术，对CRISPR RNA（crRNA）靶点区域周边的高分子量基因组DNA开展了纳米孔测序分析。