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• W2950000827 abstract "•Cas13a RNA binding and RNase activity are differentially affected by mismatches•Perfect base pairing in the guide seed region is required for stable RNA binding•RNA mismatches in a different guide region result in binding, but not RNase activity•The RNA binding and RNase activities of Cas13a can be decoupled CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development. CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development. Prokaryotic adaptive immune systems use CRISPRs and Cas proteins for RNA-guided cleavage of foreign genetic elements (Mohanraju et al., 2016Mohanraju P. Makarova K.S. Zetsche B. Zhang F. Koonin E.V. van der Oost J. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems.Science. 2016; 353: aad5147Crossref PubMed Scopus (372) Google Scholar, Wright et al., 2016Wright A.V. Nuñez J.K. Doudna J.A. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering.Cell. 2016; 164: 29-44Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar). Type VI CRISPR-Cas systems include a single protein, Cas13 (formerly C2c2), that when assembled with a CRISPR-RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1138) Google Scholar, East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Shmakov et al., 2015Shmakov S. Abudayyeh O.O. Makarova K.S. Wolf Y.I. Gootenberg J.S. Semenova E. Minakhin L. Joung J. Konermann S. Severinov K. et al.Discovery and functional characterization of diverse class 2 CRISPR-Cas systems.Mol. Cell. 2015; 60: 385-397Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar, Smargon et al., 2017Smargon A.A. Cox D.B. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28.Mol. Cell. 2017; 65: 618-630Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Yan et al., 2018Yan W.X. Chong S. Zhang H. Makarova K.S. Koonin E.V. Cheng D.R. Scott D.A. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein.Mol. Cell. 2018; 70: 327-339Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Cas13 proteins are classified into distinct subfamilies (Cas13a–Cas13d), and all Cas13 proteins studied to date possess two enzymatically distinct RNase activities that are required for optimal interference (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1138) Google Scholar, East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Smargon et al., 2017Smargon A.A. Cox D.B. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28.Mol. Cell. 2017; 65: 618-630Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Yan et al., 2018Yan W.X. Chong S. Zhang H. Makarova K.S. Koonin E.V. Cheng D.R. Scott D.A. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein.Mol. Cell. 2018; 70: 327-339Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). First, upon binding a precursor CRISPR-RNA (pre-crRNA), Cas13 cleaves within the crRNA direct repeat in a pre-crRNA array to form mature Cas13-crRNA complexes (East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, East-Seletsky et al., 2017East-Seletsky A. O’Connell M.R. Burstein D. Knott G.J. Doudna J.A. RNA targeting by functionally orthogonal type VI-A CRISPR-Cas enzymes.Mol. Cell. 2017; 66: 373-383Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Smargon et al., 2017Smargon A.A. Cox D.B. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28.Mol. Cell. 2017; 65: 618-630Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Yan et al., 2018Yan W.X. Chong S. Zhang H. Makarova K.S. Koonin E.V. Cheng D.R. Scott D.A. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein.Mol. Cell. 2018; 70: 327-339Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Second, binding of an RNA target complementary to the crRNA (henceforth referred to as an activator-RNA) triggers Cas13 to cleave RNA nonspecifically by activating the enzyme’s two higher eukaryotes and prokaryotes nucleotide binding (HEPN) domains to form a single composite RNase active site (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1138) Google Scholar, East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Liu et al., 2017aLiu L. Li X. Ma J. Li Z. You L. Wang J. Wang M. Zhang X. Wang Y. The molecular architecture for RNA-guided RNA cleavage by Cas13a.Cell. 2017; 170: 714-726Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, Liu et al., 2017bLiu L. Li X. Wang J. Wang M. Chen P. Yin M. Li J. Sheng G. Wang Y. Two distant catalytic sites are responsible for C2c2 RNase activities.Cell. 2017; 168: 121-134Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, Smargon et al., 2017Smargon A.A. Cox D.B. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28.Mol. Cell. 2017; 65: 618-630Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Yan et al., 2018Yan W.X. Chong S. Zhang H. Makarova K.S. Koonin E.V. Cheng D.R. Scott D.A. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein.Mol. Cell. 2018; 70: 327-339Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). This HEPN-activated conformation of Cas13 is a general nuclease, which is capable of cleaving either the RNA molecule that it was activated by (cis-cleavage) or any other RNA molecule that it happens to encounter (trans- or collateral cleavage). All Cas13 proteins characterized to date exhibit these behaviors (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1138) Google Scholar, East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, East-Seletsky et al., 2017East-Seletsky A. O’Connell M.R. Burstein D. Knott G.J. Doudna J.A. RNA targeting by functionally orthogonal type VI-A CRISPR-Cas enzymes.Mol. Cell. 2017; 66: 373-383Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, Gootenberg et al., 2017Gootenberg J.S. Abudayyeh O.O. Lee J.W. Essletzbichler P. Dy A.J. Joung J. Verdine V. Donghia N. Daringer N.M. Freije C.A. et al.Nucleic acid detection with CRISPR-Cas13a/C2c2.Science. 2017; 356: 438-442Crossref PubMed Scopus (1473) Google Scholar, Gootenberg et al., 2018Gootenberg J.S. Abudayyeh O.O. Kellner M.J. Joung J. Collins J.J. Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6.Science. 2018; 360: 439-444Crossref PubMed Scopus (1056) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Smargon et al., 2017Smargon A.A. Cox D.B. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28.Mol. Cell. 2017; 65: 618-630Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Yan et al., 2018Yan W.X. Chong S. Zhang H. Makarova K.S. Koonin E.V. Cheng D.R. Scott D.A. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein.Mol. Cell. 2018; 70: 327-339Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The RNA-activated RNA cleavage behavior of Cas13 provides a mechanism for RNA detection and diagnostic applications (East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, Gootenberg et al., 2017Gootenberg J.S. Abudayyeh O.O. Lee J.W. Essletzbichler P. Dy A.J. Joung J. Verdine V. Donghia N. Daringer N.M. Freije C.A. et al.Nucleic acid detection with CRISPR-Cas13a/C2c2.Science. 2017; 356: 438-442Crossref PubMed Scopus (1473) Google Scholar, Gootenberg et al., 2018Gootenberg J.S. Abudayyeh O.O. Kellner M.J. Joung J. Collins J.J. Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6.Science. 2018; 360: 439-444Crossref PubMed Scopus (1056) Google Scholar), as well as RNA targeting in heterologous systems (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (959) Google Scholar, Aman et al., 2018Aman R. Ali Z. Butt H. Mahas A. Aljedaani F. Khan M.Z. Ding S. Mahfouz M. RNA virus interference via CRISPR/Cas13a system in plants.Genome Biol. 2018; 19: 1Crossref PubMed Scopus (231) Google Scholar, Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (861) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Versions of Cas13 in which the HEPN domains have been catalytically inactivated (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1138) Google Scholar, East-Seletsky et al., 2016East-Seletsky A. O’Connell M.R. Knight S.C. Burstein D. Cate J.H. Tjian R. Doudna J.A. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.Nature. 2016; 538: 270-273Crossref PubMed Scopus (588) Google Scholar, Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, Smargon et al., 2017Smargon A.A. Cox D.B. Pyzocha N.K. Zheng K. Slaymaker I.M. Gootenberg J.S. Abudayyeh O.A. Essletzbichler P. Shmakov S. Makarova K.S. et al.Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28.Mol. Cell. 2017; 65: 618-630Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Yan et al., 2018Yan W.X. Chong S. Zhang H. Makarova K.S. Koonin E.V. Cheng D.R. Scott D.A. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein.Mol. Cell. 2018; 70: 327-339Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) (deactivated Cas13 [dCas13]) have shown themselves to be useful as programmable RNA binding proteins for RNA imaging (Abudayyeh et al., 2017Abudayyeh O.O. Gootenberg J.S. Essletzbichler P. Han S. Joung J. Belanto J.J. Verdine V. Cox D.B.T. Kellner M.J. Regev A. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (959) Google Scholar), RNA editing (Cox et al., 2017Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027Crossref PubMed Scopus (861) Google Scholar), and regulating specific transcripts RNA in other ways (e.g., regulating pre-mRNA splicing) (Konermann et al., 2018Konermann S. Lotfy P. Brideau N.J. Oki J. Shokhirev M.N. Hsu P.D. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.Cell. 2018; 173: 665-676Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). However, the relationships between crRNA: activator-RNA sequence complementarity and Cas13 binding specificity versus HEPN-nuclease activation have not been determined; based on the behavior exhibited by other CRISPR-Cas systems (for review, see Jackson et al., 2017Jackson R.N. van Erp P.B. Sternberg S.H. Wiedenheft B. Conformational regulation of CRISPR-associated nucleases.Curr. Opin. Microbiol. 2017; 37: 110-119Crossref PubMed Scopus (33) Google Scholar), it is likely that these relationships are complex. We used a combination of library-based, high-throughput RNA binding assays and biochemical experiments to interrogate the effects of mismatches on both activities of Cas13a from Leptotrichia buccalis (Lbu), enabling us to determine the relationship between activator-RNA recognition specificity and HEPN-nuclease activation. We find that both the number and the distribution of mismatches between crRNA and activator-RNA affect Lbu-Cas13a binding affinity and HEPN-nuclease activity. Mismatches in the middle seed region (positions 9–12) of the crRNA spacer result in the largest defect in binding affinity. In contrast, mismatches elsewhere in the crRNA result in only small reductions of binding affinity. Surprisingly, although targets with mismatches at positions 5–8 of the crRNA spacer bind with moderate to high affinity, these targets fail to activate Lbu-Cas13a HEPN-nuclease activity. Conversely, a separate subset of mismatched, weakly bound activator-RNAs was able to maximally activate Cas13a RNase activity. These results show that activator-RNA binding and HEPN-nuclease activation can be decoupled in Lbu-Cas13a, revealing a mechanism that is not reflected by simple activator-RNA binding affinity but instead is a complex specificity landscape that enables Lbu-Cas13a to distinguish between complementary and mismatched RNA transcripts before crRNA-guided RNase activation, all while allowing for relaxed RNA sequence specificity. Altogether, we show that the accuracies of activator-RNA binding and RNA-activated RNA cleavage by Cas13a enzymes are different, which has implications for understanding the role of Cas13a in bacterial immunity, as well as the development of Cas13a as a high-fidelity RNA-targeting tool. To determine the effects of individual crRNA:activator-RNA mismatches on the binding interaction with Lbu-Cas13a, we designed an in vitro library-based assay to assess binding mismatch tolerances and the effects of sequence variation outside the Cas13a:crRNA binding site. Our experimental design included two target RNA sequence libraries that were mutagenized in the same manner (Figure 1A) (see Experimental Procedures), but only one crRNA was used in any given binding experiment, allowing us to distinguish between weak crRNA-dependent target binding and crRNA-independent nonspecific binding. The target RNA libraries were mixed and incubated separately with two concentrations of biotinylated HEPN-nuclease deactivated Lbu-Cas13a (Lbu-dCas13a) protein (Figure S1A) (see Experimental Procedures) complexed with crRNA. After incubation, bound RNAs were eluted and subjected to Illumina-based sequencing (Figure 1B; Table S1). Controls were conducted to determine input library distribution (Figure 1C) and correct for any contribution of apo-Lbu-dCas13a background RNA binding (Figures S1B–S1K). We developed a computational pipeline to analyze our sequencing data (see Experimental Procedures) that first removes PCR duplicates (by counting unique molecular identifier sequences) and then counts the number of observations for each library variant in a given sample. For each variant, we identified the input sequence from which it was derived by Hamming distance and recorded its relative abundance in a sample. After applying a threshold to filter out low-abundance library members (fewer than 20 unique counts in each of the 3 replicates for any given condition), data from the remaining ∼18,000 unique sequences were highly reproducible across replicates, with Pearson R2 > = 0.85 in all cases (Table S1). To identify the crRNA-dependent signal from our data, we calculated fold change in abundance between pull-downs with crRNA-bound Lbu-dCas13a relative to either the apo-Lbu-dCas13a (i.e., no crRNA is present) pull-down or the initial input library. Calculating fold change in abundance relative to the apo-Lbu-dCas13a pull-down isolated the crRNA-dependent signal, whereas calculating fold change relative to the input library alone was skewed by the contribution of apo-Lbu-dCas13a (presumably from incompletely crRNA-loaded apo-Lbu-dCas13a present in the sample). Both scatter and violin plots of this fold change for each of the different crRNA-containing samples (X and Y) showed that target sequences that were strongly enriched by Lbu-dCas13a:crRNA-X were depleted by Lbu-dCas13a:crRNA-Y, and vice versa (Figures 1D, 1E, and S1D–S1K). In each case, the enriched and depleted sequences corresponded to the on-target and off-target activator-RNA libraries, respectively. This clear signal of orthogonality indicated that the sequence enrichment and depletion behavior we observed is crRNA dependent (Figures 1D, 1E, and S1D–S1K). We calculated the relative enrichment of target RNA library members with exactly one mismatch to each crRNA-guided Lbu-dCas13a sample, revealing several interesting observations (Figures 2A, 2B, and S2). First, as expected, the magnitude of enrichment for each single-mismatch member within each targeted library was highly dependent on the position of the single mismatch within the crRNA-targeting region but largely independent of which nucleotide was present on the target at the mismatched site. Second, we observed that single-mismatch members were significantly less enriched (see Experimental Procedures) when their mismatches occurred within a contiguous stretch of nucleotides in crRNA-X positions 9–14 compared to all other positions, with the effect most pronounced at high Lbu-dCas13a:crRNA concentration (Figures 2A and S2). This suggests that single base pair mismatches in positions 9–14 can significantly decrease Lbu-dCas13a affinity for activator-RNA. Conversely, library members with single base pair mismatches outside these regions (i.e., in guide RNA sequence positions 1–5 and 15–20) are enriched as much as or even more than the perfectly complementary target RNA, indicating that single mismatches in these positions in the target RNA:crRNA duplex have little to no effect on the overall binding affinity relative to a perfectly complementary target. Unexpectedly in some cases, they may even promote tighter binding than observed for perfectly complementary activator-RNA (Figure 2A). A qualitatively similar and statistically significant effect is seen when the RNA-Y library is targeted by Lbu-dCas13a:crRNA-Y, where single mismatches in positions 9–11 of the crRNA spacer are significantly less tolerated (Figures 2B and S2). The difference observed in the overall magnitude of mismatch sensitivity when comparing RNA-X and RNA-Y target libraries could be due to crRNA and target RNA sequence composition, the secondary structure propensity of the crRNA and/or target RNA, and the overall efficiency of the sample recovery. We also calculated the relative enrichment of activator-RNA library members with two mismatches and visualized the resulting data using a heatmap (Figures 2C and 2D). This analysis has the potential to show interactions between different positions of mismatches: it can uncover combinations of two mismatches that act constructively to increase the binding defect (when compared to either singly mismatched target) or pairs of mismatches whose effects compensate for each other, leading to tighter binding than that observed for either of the singly mismatched targets alone. In both the RNA-X and the RNA-Y sequences, we clearly identify a central mismatch-sensitive region (positions 9–14 for RNA-X and positions 9–11 for RNA-Y), where pairs of mismatches show even weaker binding affinity (when compared to single mismatches) (Figures 2C, 2D, and S3). Although single mismatches at positions 5–8 show moderate binding defects, we find that mismatches in these positions are able to somewhat compensate for the loss of binding affinity caused by mismatches at positions 9–12 (Figure 2C). These observations again suggest that the effect of individual mismatches on binding may not be additive when in combination, hinting that complex contributions from individual and sets of base-pairing interactions influence overall affinity for activator-RNAs. Finally, we found little evidence that sequence elements outside the crRNA:target RNA duplex (termed the protospacer-flanking motif, or PFS) (Abudayyeh et al., 2016Abudayyeh O.O. Gootenberg J.S. Konermann S. Joung J. Slaymaker I.M. Cox D.B. Shmakov S. Makarova K.S. Semenova E. Minakhin L. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353: aaf5573Crossref PubMed Scopus (1138) Google Scholar, Liu et al., 2017bLiu L. Li X. Wang J. Wang M. Chen P. Yin M. Li J. Sheng G. Wang Y. Two distant catalytic sites are responsible for C2c2 RNase activities.Cell. 2017; 168: 121-134Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) contribute to crRNA-guided Lbu-dCas13a binding to target sequences (see the lack of significant nucleotide-specific depletion in the flanking regions in Figures 2A, 2B, and S3). Specifically, the prediction from homologous Cas13a proteins and crRNAs is that Lbu-Cas13a:crRNA prefers a H at position −1 (i.e., the last position within the left flanking sequence), and the presence of a guanine is thought to inhibit Cas13a RNA cleavage. Because we do not observe depletion of library members that contain a guanine in this position, we conclude that at least for Lbu-Cas13a, a H PFS isn’t required for optimal activator-RNA binding. We independently validated the mismatch-sensitivity profiles by employing fluorescence anisotropy assays to measure target RNA binding to Lbu-dCas13a. We paired activator-RNAs containing sets of four contiguous nucleotide mismatches with an otherwise complementary crRNA (Figure 3A), tiled across the 20-nt guide sequence (Figure 3B). For three of five of these activator-RNAs (g1-4MM, g13-16MM, and g17-20MM), the affinity of the crRNA-Cas13a interaction decreased by 6- to 8-fold compared to the interaction with a perfectly complementary activator-RNA (noMM) (Figures 3B and 3C). As expected from the high-throughput binding experiment, mismatches in nucleotide positions 9–12 of the crRNA spacer (activator-RNA g9-12MM) led to the largest binding defect, a 47-fold decrease in binding affinity compared to a fully complementary single-stranded RNA (ssRNA). Because our sequencing assay hinted that pairs of mismatches at positions 5–8 (for RNA-X) could somehow compensate for the binding defect seen for the analogous singly mismatched sequences, we were interested in understanding the effect of four contiguous mismatches at positions 5–8 (g5-8MM). To our surprise, we noticed that in comparison to other sets of mismatches, g5-8MM exhibited far more moderate ∼2-fold reduction in binding affinity relative to the noMM target in our fluorescence anisotropy assay (Figures 3B and 3C). We also noticed that the overall change in the fluorescence anisotropy signal between unbound and fully bound activator-RNA was substantially lower (at least 2-fold) for g5-8MM compared to noMM activator-RNAs (Figure 3B, binding curves marked with an asterisk). This atypical effect was reproducible across three RNA sequences (Figure S4A). Thus, we speculated that the g5-8MM result might be artifactual, possibly a result of the 3′ fluorophore exhibiting significantly more rotational flexibility upon binding, leading to an inaccurate estimation of binding affinity. To disentangle this potentially confounding effect from the binding behavior of targets with mismatches at positions 5–8, we performed (fluorescence independent) filter binding e" @default.
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• W2950000827 date "2018-07-01" @default.
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• W2950000827 title "RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a" @default.
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• W2950000827 doi "https://doi.org/10.1016/j.celrep.2018.06.105" @default.
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