{"doi":"10.1101/2023.10.05.560533","title":"RNA structure modulates Cas13 activity and enables mismatch detection","abstract":"Abstract The RNA-targeting CRISPR nuclease Cas13 has emerged as a powerful tool for applications ranging from nucleic acid detection to transcriptome engineering and RNA imaging 1–6 . Cas13 is activated by the hybridization of a CRISPR RNA (crRNA) to a complementary single-stranded RNA (ssRNA) protospacer in a target RNA 1,7 . Though Cas13 is not activated by double-stranded RNA (dsRNA) in vitro , it paradoxically demonstrates robust RNA targeting in environments where the vast majority of RNAs are highly structured 2,8 . Understanding Cas13’s mechanism of binding and activation will be key to improving its ability to detect and perturb RNA; however, the mechanism by which Cas13 binds structured RNAs remains unknown 9 . Here, we systematically probe the mechanism of LwaCas13a activation in response to RNA structure perturbations using a massively multiplexed screen. We find that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: structure in the protospacer region competes with the crRNA and can be disrupted via a strand-displacement mechanism, while structure in the region 3’ to the protospacer has an allosteric inhibitory effect. We leverage the kinetic nature of the strand displacement process to improve Cas13-based RNA detection, enhancing mismatch discrimination by up to 50-fold and enabling sequence-agnostic mutation identification at low (<1%) allele frequencies. Our work sets a new standard for CRISPR-based nucleic acid detection and will enable intelligent and secondary-structure-guided target selection while also expanding the range of RNAs available for targeting with Cas13.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":390828,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9542,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1163073,"name":"Benjamin B. Larsen","orcid":null,"position":1,"is_corresponding":false},{"id":1163074,"name":"Owen R. S. Dunkley","orcid":null,"position":2,"is_corresponding":false},{"id":554169,"name":"Aartjan J.W. te Velthuis","orcid":"0000-0002-5129-3953","position":3,"is_corresponding":false},{"id":35849,"name":"Cameron Myhrvold","orcid":"0000-0002-8971-184X","position":4,"is_corresponding":false},{"id":313410,"name":"Ofer Kimchi","orcid":"0000-0002-0801-0727","position":0,"is_corresponding":true}],"reference_count":46,"raw_metadata":null,"created_at":"2026-07-19T01:18:37.743462Z","pmid":"37987004","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}