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

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. Analysis of indel frequencies by short-read Illumina sequencing of PCR amplicons surrounding genomic sites targeted by Cas12a variant fusion proteins using CRISPR RNAs (crRNA).

多重遗传扰动（multiplexed genetic perturbations）是解析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，且在池化筛选实验中更高效地实现非编码调控元件的扰动。但当前的CRISPRi池化筛选方法仅能实现单个细胞靶向1~3个基因组位点，存在应用局限。本研究构建了一种来自氨基酸球菌属（Acidaminococcus）的Cas12a（AsCas12a）变体——多重转录干扰型Cas12a（multiplexed transcriptional interference AsCas12a, multiAsCas12a），其携带R1226A突变，可通过DNA切口形成稳定的核糖核蛋白-DNA复合物。相较于核酸酶失活型AsCas12a-KRAB融合蛋白，multiAsCas12a-KRAB融合蛋白可显著增强CRISPRi活性，通常能恢复与后者联用会失活的慢病毒递送CRISPR RNA（CRISPR RNAs, crRNA）的活性。multiAsCas12a-KRAB可在高通量池化筛选中支持基于6重crRNA阵列的CRISPRi操作。借助multiAsCas12a-KRAB系统，本研究在人类细胞中成功鉴定增强子元件，并解析了顺式调控元件的组合功能。上述研究成果构建了一套群组测试框架，可高效筛选大量染色质扰动组合，用于生物学发现与工程化改造。本研究通过对使用CRISPR RNA（crRNA）的Cas12a变体融合蛋白所靶向的基因组位点侧翼的PCR扩增子进行短读长Illumina测序，分析插入缺失（insertion-deletion, indel）频率。