美国加州大学旧金山分校L. A. Gilbert课题组开发出用于高阶组合染色质扰动的工程化CRISPR-Cas12a。2024年5月17日，《自然—生物技术》杂志在线发表了这项成果。

研究人员设计了一种氨基酸球菌属Cas12a（AsCas12a）变体——多重转录干扰AsCas12a（multiAsCas12a），它结合了R1226A（这是一种突变，可通过DNA连接稳定核糖核蛋白-DNA复合物）。与DNA酶活缺失AsCas12a-KRAB融合体相比，multiAsCas12a-KRAB融合体提高了CRISPR干扰（CRISPRi）的活性，往往能挽救慢病毒递送的CRISPR RNA（crRNA）的活性，因为后者在使用时没有活性。

利用multiAsCas12a-KRAB，研究人员发现了增强子元件，并剖析了人类细胞中顺式调节元件的组合功能。这些结果建立了一个群组测试框架，可以高效地调查染色质扰动的多种组合，用于生物发现和工程学研究。

据了解，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形成抑制性染色质可避免基因毒性，而且在集合试验中对非编码调控元件的扰动更为有效。然而，目前的CRISPRi联合筛选方法仅限于针对每个细胞的一到三个基因组位点。

附：英文原文

Title: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations

Author: Hsiung, C. C.-S., Wilson, C. M., Sambold, N. A., Dai, R., Chen, Q., Teyssier, N., Misiukiewicz, S., Arab, A., OLoughlin, T., Cofsky, J. C., Shi, J., Gilbert, L. A.

Issue&Volume: 2024-05-17

Abstract: 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. 

DOI: 10.1038/s41587-024-02224-0

Source: https://www.nature.com/articles/s41587-024-02224-0