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CRISPR Screening Approach for Mapping Genetic Interactions Uncovers Masked Phenotypes

NEW YORK – A team led by researchers at the University of Toronto has developed a CRISPR-based screening system that multiplexes Cas9 and Cas12a, which they've used to systemically map genetic interactions and interrogate the functions of large genomic segments in mammalian cells.

The system, which also involves machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs, is named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), the researchers wrote in a study describing the work on Monday in Nature Biotechnology. In their experiments, they found that the combination of Cas9 and Cas12a outperformed genetic screens using either Cas9 or Cas12a editing alone.

Applying CHyMErA to the ablation of mammalian paralog gene pairs revealed extensive genetic interactions and uncovered phenotypes normally masked by functional redundancy, the researchers wrote. Further, when they applied CHyMErA in a chemogenetic interaction screen, they were able to identify genes that affect cell growth in response to inhibition of the mTOR pathway. And by systematically targeting thousands of alternative splicing events, CHyMErA also identified exons underlying human cell line fitness.

"CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons," the authors wrote.

CHyMErA uses coexpression of Streptococcus pyogenes Cas9 (SpCas9) and Lachnospiraceae bacterium Cas12a (LbCas12a), together with hybrid guide RNAs (hgRNAs) generated from fusions of Cas9 and Cas12a gRNAs expressed from a single promoter. Using CHyMErA, the team performed screens with optimized hgRNA libraries targeting 672 human paralog pairs representing more than 90 percent of predicted duplicate paralog genes in the human genome, explored chemogenetic interactions in the mTOR pathway, and interrogated the functions of 2,157 alternative cassette exons in cell fitness.

"These screens demonstrate a previously unappreciated degree of complexity of [genetic interactions (GIs)] among paralogous genes, reveal new chemical GIs, and identify numerous alternative exons that impact cell growth," the researchers said.

CHyMErA was also able to detect digenic interactions. To assess the efficacy of the multisite-targeting capacity of CHyMErA for mapping GIs, the researchers used optimized hgRNAs to target pairs of genes involved in a small number of known digenic interactions. This screen detected expected GIs between TP53 and its negative regulators MDM4 and MDM2 in RPE1 cells, which express wild-type TP53. However, these interactions were not detected in HAP1 cells, which harbor an expressed but inactive mutant version of TP53. CHyMErA also captured known negative GIs between MCL1 and BCL2L1, and between KDM6B and BRD4, the researchers wrote, demonstrating its utility in mapping digenic interactions in mammalian cells.

"The combination of Cas9 and Cas12a systems leverages twice the number of possible targeting sites in comparison to the use of either enzyme alone — that is, the human genome contains 227.3 million Cas9 and 207.6 million Cas12a candidate PAM target sites," the authors concluded. "Engineering Cas9, Cas12a, or other Cas enzymes for increased editing efficiency and target specificity is expected to further expand the targeting landscape of the CHyMErA system."

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