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Optimization of AsCas12a for combinatorial genetic screens in human cells

Kendall R Sanson, Peter C DeWeirdt, Annabel K Sangree, Ruth E Hanna, Mudra Hegde, Teng Teng, Samantha M Borys, Christine Strand, J Keith Joung, Benjamin P Kleinstiver, Xuewen Pan, Alan Huang, View ORCID ProfileJohn G Doench
doi: https://doi.org/10.1101/747170
Kendall R Sanson
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Peter C DeWeirdt
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Annabel K Sangree
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Ruth E Hanna
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Mudra Hegde
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Teng Teng
2Tango Therapeutics, 100 Binney St., Cambridge, MA
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Samantha M Borys
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Christine Strand
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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J Keith Joung
3Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA
4Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA
5Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA
6Department of Pathology, Harvard Medical School, Boston, MA, USA
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Benjamin P Kleinstiver
6Department of Pathology, Harvard Medical School, Boston, MA, USA
7Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114
8Department of Pathology, Massachusetts General Hospital, Boston, MA 02114
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Xuewen Pan
2Tango Therapeutics, 100 Binney St., Cambridge, MA
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Alan Huang
2Tango Therapeutics, 100 Binney St., Cambridge, MA
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John G Doench
1Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA
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  • ORCID record for John G Doench
  • For correspondence: jdoench@broadinstitute.org
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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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Posted August 28, 2019.
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Optimization of AsCas12a for combinatorial genetic screens in human cells
Kendall R Sanson, Peter C DeWeirdt, Annabel K Sangree, Ruth E Hanna, Mudra Hegde, Teng Teng, Samantha M Borys, Christine Strand, J Keith Joung, Benjamin P Kleinstiver, Xuewen Pan, Alan Huang, John G Doench
bioRxiv 747170; doi: https://doi.org/10.1101/747170
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Optimization of AsCas12a for combinatorial genetic screens in human cells
Kendall R Sanson, Peter C DeWeirdt, Annabel K Sangree, Ruth E Hanna, Mudra Hegde, Teng Teng, Samantha M Borys, Christine Strand, J Keith Joung, Benjamin P Kleinstiver, Xuewen Pan, Alan Huang, John G Doench
bioRxiv 747170; doi: https://doi.org/10.1101/747170

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