PT - JOURNAL ARTICLE AU - Kendall R Sanson AU - Peter C DeWeirdt AU - Annabel K Sangree AU - Ruth E Hanna AU - Mudra Hegde AU - Teng Teng AU - Samantha M Borys AU - Christine Strand AU - J Keith Joung AU - Benjamin P Kleinstiver AU - Xuewen Pan AU - Alan Huang AU - John G Doench TI - Optimization of AsCas12a for combinatorial genetic screens in human cells AID - 10.1101/747170 DP - 2019 Jan 01 TA - bioRxiv PG - 747170 4099 - http://biorxiv.org/content/early/2019/08/28/747170.short 4100 - http://biorxiv.org/content/early/2019/08/28/747170.full AB - 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.