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Optimization of AsCas12a for combinatorial genetic screens in human cells

Nature Biotechnology volume 39, pages 94–104 (2021)Cite this article

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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.

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Fig. 1: Optimization of AsCas12a for pooled screens.
Fig. 2: On-target design rules for enCas12a.
Fig. 3: Prediction of off-target activity for AsCas12a.
Fig. 4: Development of alternative DR sequences for multiplexing with AsCas12a.
Fig. 5: Validation of AsCas12a performance with synthetic lethal gene pairs.
Fig. 6: Combinatorial screen identifies a new synthetic lethality in apoptotic genes.
Fig. 7: Genome-wide libraries for enCas12a.

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Data availability

The read counts for all screening data and subsequent analyses are provided as Supplementary Data and are available on the Sequence Read Archive, accession no. SRP228317.

Code availability

All customized code used for analysis and notebooks is available on GitHub: https://github.com/PeterDeWeirdt.

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Acknowledgements

We thank A. Goodale, B. Fritchman and X. Yang for producing guide libraries and lentivirus; O. Bare, M. Macaluso and Y. Lee for logistics support; C. Petersen for assistance with screens; M. Greene, A. Brown, D. Alan, M. Tomko and T. Green for software engineering support; the Broad Institute Genomics Platform Walk-up Sequencing group for Illumina sequencing; the Broad Institute Flow Cytometry Facility for assistance with gating strategy; and the Functional Genomics Consortium for funding support.

Author information

Author notes

  1. These authors contributed equally: Peter C. DeWeirdt, Kendall R. Sanson, Annabel K. Sangree, Mudra Hegde.

Authors and Affiliations

  1. Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Peter C. DeWeirdt, Kendall R. Sanson, Annabel K. Sangree, Mudra Hegde, Ruth E. Hanna, Marissa N. Feeley, Audrey L. Griffith, Samantha M. Borys, Christine Strand & John G. Doench

  2. Tango Therapeutics, Cambridge, MA, USA

    Teng Teng, Xuewen Pan & Alan Huang

  3. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA

    J. Keith Joung

  4. Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA

    J. Keith Joung

  5. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA

    J. Keith Joung

  6. Department of Pathology, Harvard Medical School, Boston, MA, USA

    J. Keith Joung & Benjamin P. Kleinstiver

  7. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Benjamin P. Kleinstiver

  8. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Benjamin P. Kleinstiver

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Contributions

X.P., A.H. and J.G.D. conceived the study. J.G.D. supervised the project. T.T. and X.P. generated the 2xNLS-Cas12a construct. B.P.K. and J.K.J. provided the sequence of enAsCas12a in advance of publication. M.H., P.C.D. and R.E.H. designed the libraries. K.R.S., A.K.S., C.S., S.M.B., M.N.F. and A.L.G. executed the genetic screens. P.C.D., M.H., K.R.S. and J.G.D. performed the analyses. P.C.D., A.K.S., K.R.S. and M.H. created the visualizations. P.C.D. and M.H. curated the data. P.C.D., A.K.S., K.R.S., R.E.H. and J.G.D. wrote the manuscript.

Corresponding author

Correspondence to John G. Doench.

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Competing interests

J.G.D. consults for Foghorn Therapeutics, Maze Therapeutics, Merck, Agios and Pfizer; he also consults for and has equity in Tango Therapeutics. B.P.K. is a scientific advisor to Avectas. T.T., X.P. and A.H. are employees of Tango Therapeutics. J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, Transposagen Biopharmaceuticals and Verve Therapeutics (formerly known as Endcadia); his interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. He is a member of the Board of Directors of the American Society of Gene and Cell Therapy. J.K.J. and B.P.K. are co-inventors on various patents and patent applications that describe gene editing and epigenetic editing technologies, including the enhanced Cas12a variant used in the present study. A patent application has been filed on the basis of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–16, Supplementary Tables 1–3 and Supplementary Note 1.

Reporting Summary

Supplementary Data 1

Read counts from on-target tiling libraries for comparing the performance of 1xNLS-Cas12a, 2xNLS-Cas12a, enCas12a and SpCas9.

Supplementary Data 2

Read counts for the PAM tiling library to define the preferences for enCas12a.

Supplementary Data 3

Read counts from mismatch libraries for off-target effects of 2xNLS-Cas12a and enCas12a.

Supplementary Data 4

Read counts from assays to identify alternative DR sequences.

Supplementary Data 5

Read counts for the synthetic lethality library screened with 2xNLS-Cas12a and enCas12a.

Supplementary Data 6

Read counts for the apoptosis combinatorial library screened with enCas12a.

Supplementary Data 7

Read counts for the Humagne set A library.

Supplementary Data 8

Read counts for the Humagne set B library.

Supplementary Data 9

Read counts for the Brunello library.

Supplementary Data 10

Precision-recall analysis for essential genes for Cas9 and Cas12a genome-wide libraries. First tab contains guide-level recall at 95% precision; second tab contains gene-level recall at 95% precision after averaging together all guides targeting the same gene. For the Brunello and Humagne screens, recall is calculated both for individual replicates and for merged replicates. For the Avana dataset, we used the cell lines that had exactly 2 replicates (207 of 341). For GeCKOv2, we used the cell lines that had exactly 4 replicates (29 of 33).

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DeWeirdt, P.C., Sanson, K.R., Sangree, A.K. et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat Biotechnol 39, 94–104 (2021). https://doi.org/10.1038/s41587-020-0600-6

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