Optimization of AsCas12a for combinatorial genetic screens in human cells

Cas12a enzymes have attractive properties for scalable delivery of multiplexed perturbations, yet widespread usage has lagged behind Cas9-based strategies. Here we describe the optimization of Cas12a from Acidaminococcus (AsCas12a) for use in pooled genetic screens in human cells. By assaying the activity of thousands of guides, we confirm on-target design rules and extend them to an enhanced activity variant, enAsCas12a. We also develop the first comprehensive set of off-target rules for Cas12a, and demonstrate that we can predict and exclude promiscuous guides. Finally, to enable efficient higher-order multiplexing via lentiviral delivery, we screen thousands of direct repeat variants and identify 38 that outperform the wildtype sequence. We validate this optimized AsCas12a toolkit by targeting 12 synthetic lethal gene pairs with up to 400 guide pairs each, and demonstrate effective triple knockout via flow cytometry. These results establish AsCas12a as a robust system for combinatorial applications of CRISPR technology.

Cas12a酶（Cas12a）具备实现规模化多重基因扰动递送的优良特性，但其实际应用的普及程度却落后于基于Cas9的相关研究策略。本研究针对人细胞混合池遗传筛选场景，阐述了源自氨基酸球菌属的Cas12a（AsCas12a）的优化方案。通过对数千条向导RNA（guide）的活性进行检测，我们验证了Cas12a的靶向设计规则，并将该规则拓展至高活性变体enAsCas12a。本研究还首次构建了一套完整的Cas12a脱靶效应设计规则，并证实可通过该规则预测并剔除具有混杂结合活性的向导RNA。最后，为实现基于慢病毒递送的高效高阶多重编辑，我们对数千条直接重复序列变体开展筛选，最终鉴定出38条性能优于野生型序列的变体。我们通过靶向12组合成致死基因对（每组最多包含400条向导RNA对）对该优化后的AsCas12a工具包进行了验证，并借助流式细胞术证实了高效的三重基因敲除效果。上述研究结果证实AsCas12a可作为CRISPR技术组合应用的稳健实验平台。