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 one to three 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.

多重遗传扰动对于测试编码或非编码遗传元件之间的功能相互作用至关重要。与双链 DNA 切割相比，使用 CRISPR 干扰 (CRISPRi) 抑制染色质形成可避免基因毒性，并且在混合分析中更有效地干扰非编码调控元件。然而，目前的 CRISPRi 联合筛选方法仅限于针对每个细胞一到三个基因组位点。我们设计了一种氨基酸球菌Cas12a (AsCas12a) 变体，即多重转录干扰 AsCas12a (multiAsCas12a)，其中包含 R1226A，这是一种通过 DNA 切口稳定核糖核蛋白-DNA 复合物的突变。 multiAsCas12a-KRAB 融合体比 DNase 死亡的 AsCas12a-KRAB 融合体提高了 CRISPRi 活性，通常可以挽救慢病毒递送的 CRISPR RNA (crRNA) 的活性，而后者与后者一起使用时会失去活性。 multiAsCas12a-KRAB 在高通量混合筛选中使用 6 重 crRNA 阵列支持 CRISPRi。使用 multiAsCas12a-KRAB，我们发现了增强子元件并剖析了人类细胞中顺式调节元件的组合功能。这些结果实例化了一个群体测试框架，用于有效地调查生物发现和工程中染色质扰动的多种组合。