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Updated: Dec 15, 2025
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Optimization of AsCas12a for combinatorial genetic screens in human cells
Peter C DeWeirdt1, Kendall R Sanson1, Annabel K Sangree1
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
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.
Insights
Enhanced Cas12a (enAsCas12a) improves multiplexed genetic screening in human cells. This optimized toolkit offers Cas9-like performance with smaller libraries, facilitating complex genetic screens and identifying novel synthetic lethalities.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Cas12a RNA-guided endonucleases enable multiplexed genetic perturbations by processing multiple guide RNAs.
- Widespread adoption of Cas12a lags behind Cas9 due to lower activity and limited screening toolkits.
Purpose of the Study:
- Optimize enhanced Cas12a (enAsCas12a) for pooled, combinatorial genetic screens in human cells.
- Develop refined design rules and off-target prediction for Cas12a guides.
- Validate the enAsCas12a toolkit in cancer cell line screens.
Main Methods:
- Assayed thousands of guides to refine on-target and off-target design rules for enAsCas12a.
- Identified 38 direct repeat variants for improved Cas12a function.
- Screened for synthetic lethalities in OVCAR8 and A375 cancer cells using the optimized toolkit.
Main Results:
- Established robust on-target and off-target design rules for enAsCas12a.
- Discovered a synthetic lethality interaction between MARCH5 and WSB2.
- Demonstrated enAsCas12a achieves genome-wide dropout screen performance comparable to Cas9 with reduced library size.
Conclusions:
- The optimized enAsCas12a toolkit significantly enhances multiplexed genetic screening capabilities.
- This toolkit facilitates screens in challenging models due to its efficiency and reduced library requirements.
- The findings pave the way for broader application of Cas12a in functional genomics research.
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