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

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. Cas12a CRISPRi pooled screens using single-plex crRNA and 6-plex crRNA libraries in K562 cells, using cell fitness as readout.

多重遗传扰动是验证编码或非编码遗传元件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）的染色质阻抑策略可规避遗传毒性，且在混合筛选实验中更高效地扰动非编码调控元件。然而，当前CRISPRi混合筛选方法仅局限于单个细胞靶向1~3个基因组位点。本研究工程化构建了一种酸球菌Cas12a（Acidaminococcus Cas12a, AsCas12a）变体——多重转录干扰型AsCas12a（multiplexed transcriptional interference AsCas12a, multiAsCas12a），其引入了R1226A突变，该突变可通过DNA单链切口稳定核糖核蛋白-DNA复合物。相较于无DNase活性的AsCas12a-KRAB融合蛋白，multiAsCas12a-KRAB融合蛋白可提升CRISPRi活性，通常能挽救那些与前者联用时失活的慢病毒递送型CRISPR RNA（crRNA）的活性。multiAsCas12a-KRAB可支持在高通量混合筛选中使用6重crRNA阵列开展CRISPRi实验。借助multiAsCas12a-KRAB，本研究鉴定出增强子元件，并解析了人类细胞中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。本研究在K562细胞中分别使用单重crRNA与6重crRNA文库开展Cas12a CRISPRi混合筛选，以细胞适应性作为检测读端。