美国麻省理工学院和哈佛大学John G. Doench研究组发现，优化的AsCas12a可用于人细胞的组合遗传筛选。相关论文于2020年7月13日发表于《自然-生物技术》。

研究人员优化了酸性氨基球菌的Cas12a（enAsCas12a），以应用于人细胞中的组合遗传筛选。通过分析成千上万条向导RNA的活性，研究人员完善了靶点设计规则，并开发了一套全面预测脱靶效应的程序来排除混杂的向导RNA。研究还确定了38个直接重复变体，可以替代野生型序列。通过筛选OVCAR8和A375癌细胞中的合成致死率，研究人员发现MARCH5和WSB2之间的相互作用，以验证优化的AsCas12a工具包。

最后，该研究证明了enAsCas12a在全基因组筛选中可提供与Cas9相似的性能，但大大减小了文库大小，这将有助于在具有挑战性的模式生物中进行筛选。

研究人员表示，Cas12a RNA介导的核酸内切酶是可用于多重遗传干扰的潜在工具，因为它们可以处理表达为单个转录本的多条向导RNA，并随后切割靶DNA。但是，由于活性低和缺乏验证的合并筛选工具包，其应用落后于基于Cas9的遗传筛选工具。

附：英文原文

Title: Optimization of AsCas12a for combinatorial genetic screens in human cells

Author: Peter C. DeWeirdt, Kendall R. Sanson, Annabel K. Sangree, Mudra Hegde, Ruth E. Hanna, Marissa N. Feeley, Audrey L. Griffith, Teng Teng, Samantha M. Borys, Christine Strand, J. Keith Joung, Benjamin P. Kleinstiver, Xuewen Pan, Alan Huang, John G. Doench

Issue&Volume: 2020-07-13

Abstract: Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models. Improved Cas12a variants and sgRNA design rules enhance genome-wide screens.

DOI: 10.1038/s41587-020-0600-6

Source: https://www.nature.com/articles/s41587-020-0600-6