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Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening

Nature Protocols volume 16, pages 4722–4765 (2021)Cite this article

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Abstract

CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15–20 weeks.

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Fig. 1: CHyMErA combinatorial genome-editing platform.
Fig. 2: Experimental workflow of CHyMErA screens.
Fig. 3: Anticipated results for Cas9/Cas12a cell line characterization, hgRNA cloning, sequencing library production and screen performance assessment.
Fig. 4: hgRNA cloning process.
Fig. 5: Generation of NGS sequencing libraries.

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Data availability

All plasmids and CHyMErA screening libraries described in this protocol are available from Addgene (http://www.addgene.org/), and a detailed list of the respective Addgene reagent numbers is provided in the reagent section of this protocol. Published example data sets21 can be accessed via Gene Expression Omnibus GSE144281.

Code availability

Published codes for dual-targeting, genetic interaction and exon-deletion scoring are available on GitHub (https://github.com/HenryWard/chymera-scoring; https://github.com/BlencoweLab/CHyMErA_exonDelScoring)21. An accompanying protocol describes in detail computational approaches for the processing of Illumina sequencing data and the mapping of genetic interaction using CHyMErA combinatorial screens65.

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Acknowledgements

We thank J. Moffat, B. J. Blencowe for their support and members of the Moffat and Blencowe laboratories at the University of Toronto for helpful discussions. We also thank H. Ward for valuable feedback and comments. Finally, we thank all members of the Gonatopoulos-Pournatzis group for their feedback. T.G.-P. is supported by the NIH Earl Stadtman Investigator Program and the NIH Distinguished Scholars Program.

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  1. RNA Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Michael Aregger, Kun Xing & Thomas Gonatopoulos-Pournatzis

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  1. Michael Aregger
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M.A. and T.G.-P. wrote the manuscript with major contributions from K.X.

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Correspondence to Michael Aregger or Thomas Gonatopoulos-Pournatzis.

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A patent application (no. GB 1907733.8) describing the development and applications of CHyMErA is pending.

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Gonatopoulos-Pournatzis, T. et al. Nat. Biotechnol. 38, 638–648 (2020): https://doi.org/10.1038/s41587-020-0437-z

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Aregger, M., Xing, K. & Gonatopoulos-Pournatzis, T. Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening. Nat Protoc 16, 4722–4765 (2021). https://doi.org/10.1038/s41596-021-00595-1

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