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Deep learning of Cas13 guide activity from high-throughput gene essentiality screening

View ORCID ProfileJingyi Wei, View ORCID ProfilePeter Lotfy, View ORCID ProfileKian Faizi, Hugo Kitano, View ORCID ProfilePatrick D. Hsu, View ORCID ProfileSilvana Konermann
doi: https://doi.org/10.1101/2021.09.14.460134
Jingyi Wei
1Department of Bioengineering, Stanford University, Stanford, CA
2Department of Biochemistry, Stanford University, Stanford, CA
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Peter Lotfy
3Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA
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Kian Faizi
3Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA
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Hugo Kitano
4Department of Computer Science, Stanford University, Stanford, CA
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Patrick D. Hsu
5Department of Bioengineering, University of California, Berkeley, Berkeley, CA
6Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA
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  • For correspondence: silvanak@stanford.edu pdhsu@berkeley.edu
Silvana Konermann
2Department of Biochemistry, Stanford University, Stanford, CA
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  • For correspondence: silvanak@stanford.edu pdhsu@berkeley.edu
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Abstract

Transcriptome engineering requires flexible RNA-targeting technologies that can perturb mammalian transcripts in a robust and scalable manner. CRISPR systems that natively target RNA molecules, such as Cas13 enzymes, are enabling rapid progress in the investigation of RNA biology and advancement of RNA therapeutics. Here, we sought to develop a Cas13 platform for high-throughput phenotypic screening and elucidate the design principles underpinning its RNA targeting efficiency. We employed the RfxCas13d (CasRx) system in a positive selection screen by tiling 55 known essential genes with single nucleotide resolution. Leveraging this dataset of over 127,000 guide RNAs, we systematically compared a series of linear regression and machine learning algorithms to train a convolutional neural network (CNN) model that is able to robustly predict guide RNA performance based on guide sequence alone. We further incorporated secondary features including secondary structure, free energy, target site position, and target isoform percent. To evaluate model performance, we conducted orthogonal screens via cell surface protein knockdown. The final CNN model is able to predict highly effective guide RNAs (gRNAs) within each transcript with >90% accuracy in this independent test set. To provide user interpretability, we evaluate feature contributions using both integrated gradients and SHapley Additive exPlanations (SHAP). We identify a specific sequence motif at guide position 15-24 along with selected secondary features to be predictive of highly efficient guides. Taken together, we derive Cas13d guide design rules from large-scale screen data, release a guide design tool (http://RNAtargeting.org) to advance the RNA targeting toolbox, and describe a path for systematic development of deep learning models to predict CRISPR activity.

Competing Interest Statement

P.D.H. is a cofounder of Spotlight Therapeutics and Moment Biosciences and serves on the board of directors and scientific advisory boards, and is a scientific advisory board member to Vial Health and Serotiny. P.D.H. and S.K. are inventors on patents relating to CRISPR technologies.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 14, 2021.
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Deep learning of Cas13 guide activity from high-throughput gene essentiality screening
Jingyi Wei, Peter Lotfy, Kian Faizi, Hugo Kitano, Patrick D. Hsu, Silvana Konermann
bioRxiv 2021.09.14.460134; doi: https://doi.org/10.1101/2021.09.14.460134
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Deep learning of Cas13 guide activity from high-throughput gene essentiality screening
Jingyi Wei, Peter Lotfy, Kian Faizi, Hugo Kitano, Patrick D. Hsu, Silvana Konermann
bioRxiv 2021.09.14.460134; doi: https://doi.org/10.1101/2021.09.14.460134

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