Optimization of AsCas12a for combinatorial genetic screens in human cells

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

doi:10.1038/s41587-020-0600-6

GeneticsNature BiotechnologyJul 13, 2020

Peer-reviewed journal article

Optimization of AsCas12a for combinatorial genetic screens in human cells

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

Abstract

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.

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