Optimization of AsCas12a for combinatorial genetic screens in human cells.
Optimization of AsCas12a for combinatorial genetic screens in human cells.
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DOI：
10.1038/s41587-020-0600-6
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发表时间：
2021-01
影响因子：
46.9
通讯作者：
Doench JG
中科院分区：
文献类型：
--
作者：
DeWeirdt PC;Sanson KR;Sangree AK;Hegde M;Hanna RE;Feeley MN;Griffith AL;Teng T;Borys SM;Strand C;Joung JK;Kleinstiver BP;Pan X;Huang A;Doench JG
Cas12a RNA引导的内切酶是一种很有前景的多重遗传扰动工具，因为它们可以处理多个表达为单个转录本的引导RNA，并随后切割目标DNA。然而，由于活跃度低，以及缺乏经过充分验证的集合筛查工具包，它们的广泛采用落后于基于Cas9的策略。在这里，我们描述了增强型AsCas12a(EnAsCas12a)在人类细胞中用于混合、组合遗传筛选的优化。通过分析数千名导游的活跃度，我们提炼了目标上的设计规则，并开发了一套全面的非目标规则来预测和排除混杂的导游。我们还鉴定了38个可替代野生型序列的直接重复变异体。我们通过筛选OVCAR8和A375癌细胞中的合成致命性来验证我们优化的AsCas12a工具包，发现March5和WSB2之间存在相互作用。最后，我们展示了enAsCas12a在全基因组辍学筛选中的表现与Cas9相似，但大大减少了文库大小，这将有助于在具有挑战性的模型中进行筛选。改进的Cas12a变体和sgRNA设计规则增强了全基因组筛选。
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. Here we describe the optimization of enhanced AsCas12a (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.
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影响因子：
46.9
作者：
Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
通讯作者：
Joung JK
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
48
作者：
Chow, Ryan D.;Wang, Guangchuan;Chen, Sidi
通讯作者：
Chen, Sidi
影响因子：
48
作者：
Agudelo, Daniel;Duringer, Alexis;Doyon, Yannick
通讯作者：
Doyon, Yannick
影响因子：
14.9
作者：
Liu, Pengpeng;Luk, Kevin;Wolfe, Scot A.
通讯作者：
Wolfe, Scot A.