Nat Biotechnol. 2020 Jul 13. doi: 10.1038/s41587-020-0600-6. | PubMed

Peter C DeWeirdt¹, Kendall R Sanson¹, Annabel K Sangree¹, Mudra Hegde¹, Ruth E Hanna¹, Marissa N Feeley¹, Audrey L Griffith¹, Teng Teng², Samantha M Borys¹, Christine Strand¹, J Keith Joung^3,4,5,6, Benjamin P Kleinstiver^6,7,8, Xuewen Pan², Alan Huang², John G Doench⁹

1. Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
2. Tango Therapeutics, Cambridge, MA, USA.
3. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
4. Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.
5. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
6. Department of Pathology, Harvard Medical School, Boston, MA, USA.
7. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
8. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
9. Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA. jdoench@broadinstitute.org.

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.

Presented By Peter DeWeirdt