{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:10:48Z","timestamp":1768507848088,"version":"3.49.0"},"posted":{"date-parts":[[2023,8,21]]},"group-title":"Molecular Biology","reference-count":73,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,8,21]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is impeded by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead CRISPR-\n                  <jats:italic>Rfx<\/jats:italic>\n                  Cas13d RNA-targeting system (dCas13d\/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to\n                  <jats:italic>SMN2<\/jats:italic>\n                  , a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identified not only known SREs, but also a novel distal intronic splicing enhancer, which can be targeted to alter exon 7 splicing using either dCas13d\/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.\n                <\/jats:p>","DOI":"10.1101\/2023.08.21.554109","type":"posted-content","created":{"date-parts":[[2023,8,22]],"date-time":"2023-08-22T00:40:20Z","timestamp":1692664820000},"source":"Crossref","is-referenced-by-count":1,"title":["Deep screening of proximal and distal splicing-regulatory elements in a native sequence context"],"prefix":"10.64898","author":[{"given":"Yocelyn","family":"Recinos","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dmytro","family":"Ustianenko","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yow-Tyng","family":"Yeh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaojian","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martin","family":"Jacko","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lekha V.","family":"Yesantharao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiyang","family":"Wu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8310-7537","authenticated-orcid":false,"given":"Chaolin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023082413500502000_2023.08.21.554109v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/271501a0"},{"key":"2023082413500502000_2023.08.21.554109v1.2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.74.8.3171"},{"key":"2023082413500502000_2023.08.21.554109v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(77)90180-5"},{"key":"2023082413500502000_2023.08.21.554109v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/ng.259"},{"key":"2023082413500502000_2023.08.21.554109v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nature07509"},{"key":"2023082413500502000_2023.08.21.554109v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/nature08909"},{"key":"2023082413500502000_2023.08.21.554109v1.7","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.72.121801.161720"},{"key":"2023082413500502000_2023.08.21.554109v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2673"},{"key":"2023082413500502000_2023.08.21.554109v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2019.09.017"},{"key":"2023082413500502000_2023.08.21.554109v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2009.02.011"},{"key":"2023082413500502000_2023.08.21.554109v1.11","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1101135108"},{"key":"2023082413500502000_2023.08.21.554109v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/nature13908"},{"key":"2023082413500502000_2023.08.21.554109v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-020-0075-7"},{"key":"2023082413500502000_2023.08.21.554109v1.14","doi-asserted-by":"publisher","DOI":"10.3390\/jcm9062004"},{"key":"2023082413500502000_2023.08.21.554109v1.15","doi-asserted-by":"publisher","DOI":"10.31662\/jmaj.2021-0019"},{"key":"2023082413500502000_2023.08.21.554109v1.16","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.aaz6100"},{"key":"2023082413500502000_2023.08.21.554109v1.17","doi-asserted-by":"publisher","DOI":"10.4161\/rna.4.1.4535"},{"key":"2023082413500502000_2023.08.21.554109v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.100416"},{"key":"2023082413500502000_2023.08.21.554109v1.19","doi-asserted-by":"publisher","DOI":"10.1261\/rna.876308"},{"key":"2023082413500502000_2023.08.21.554109v1.20","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-018-1437-x"},{"key":"2023082413500502000_2023.08.21.554109v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2018.10.037"},{"key":"2023082413500502000_2023.08.21.554109v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/ng.3837"},{"key":"2023082413500502000_2023.08.21.554109v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2004.11.010"},{"key":"2023082413500502000_2023.08.21.554109v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2008.10.046"},{"key":"2023082413500502000_2023.08.21.554109v1.25","doi-asserted-by":"publisher","DOI":"10.1126\/science.1073774"},{"key":"2023082413500502000_2023.08.21.554109v1.26","doi-asserted-by":"publisher","DOI":"10.1101\/gad.1195304"},{"key":"2023082413500502000_2023.08.21.554109v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2006.05.008"},{"key":"2023082413500502000_2023.08.21.554109v1.28","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.09.054"},{"key":"2023082413500502000_2023.08.21.554109v1.29","doi-asserted-by":"publisher","DOI":"10.1038\/nature09000"},{"key":"2023082413500502000_2023.08.21.554109v1.30","doi-asserted-by":"publisher","DOI":"10.1126\/science.1254806"},{"key":"2023082413500502000_2023.08.21.554109v1.31","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0801692105"},{"key":"2023082413500502000_2023.08.21.554109v1.32","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M005597200"},{"key":"2023082413500502000_2023.08.21.554109v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.2699"},{"key":"2023082413500502000_2023.08.21.554109v1.34","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaf5573"},{"key":"2023082413500502000_2023.08.21.554109v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2017.04.008"},{"key":"2023082413500502000_2023.08.21.554109v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2016.12.023"},{"key":"2023082413500502000_2023.08.21.554109v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.02.033"},{"key":"2023082413500502000_2023.08.21.554109v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2018.02.028"},{"key":"2023082413500502000_2023.08.21.554109v1.39","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.10.008"},{"key":"2023082413500502000_2023.08.21.554109v1.40","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-021-01124-4"},{"key":"2023082413500502000_2023.08.21.554109v1.41","doi-asserted-by":"crossref","unstructured":"Wei, J. , Lotfy, P. , Faizi, K. , Wang, E. , Slabodkin, H. , Kinnaman, E. , Chandrasekaran, S. , Kitano, H. , Durrant, M.G. , Duffy, C.V. , et al. (2022). Deep learning and CRISPR-Cas13d ortholog discovery for optimized RNA targeting. bioRxiv.","DOI":"10.1101\/2021.09.14.460134"},{"key":"2023082413500502000_2023.08.21.554109v1.42","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0456-9"},{"key":"2023082413500502000_2023.08.21.554109v1.43","unstructured":"Wei, J. , Lotfy, P. , Faizi, K. , Kitano, H. , Hsu, P.D. , and Konermann, S. (2021). Deep learning of Cas13 guide activity from high-throughput gene essentiality screening. bioRxiv."},{"key":"2023082413500502000_2023.08.21.554109v1.44","doi-asserted-by":"crossref","unstructured":"Li, Z. , Li, Z. , Cheng, X. , Wang, X. , Ma, S. , Wang, S. , Lu, Z. , Zhang, H. , Zhao, W. , Chen, Z. , et al. (2022). Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems. bioRxiv.","DOI":"10.1101\/2022.05.14.491940"},{"key":"2023082413500502000_2023.08.21.554109v1.45","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-021-02263-9"},{"key":"2023082413500502000_2023.08.21.554109v1.46","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-36316-3"},{"key":"2023082413500502000_2023.08.21.554109v1.47","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-01011-4"},{"key":"2023082413500502000_2023.08.21.554109v1.48","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-58104-5"},{"key":"2023082413500502000_2023.08.21.554109v1.49","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaq0180"},{"key":"2023082413500502000_2023.08.21.554109v1.50","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0572-6"},{"key":"2023082413500502000_2023.08.21.554109v1.51","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2020.10.019"},{"key":"2023082413500502000_2023.08.21.554109v1.52","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-16806-4"},{"key":"2023082413500502000_2023.08.21.554109v1.53","doi-asserted-by":"crossref","unstructured":"Anderson, K.M. , Poosala, P. , Lindley, S.R. , and Anderson, D.M . (2019). Targeted Cleavage and Polyadenylation of RNA by CRISPR-Cas13. bioRxiv.","DOI":"10.1101\/531111"},{"key":"2023082413500502000_2023.08.21.554109v1.54","doi-asserted-by":"publisher","DOI":"10.1038\/nature24049"},{"key":"2023082413500502000_2023.08.21.554109v1.55","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2006617117"},{"key":"2023082413500502000_2023.08.21.554109v1.56","doi-asserted-by":"publisher","DOI":"10.1101\/2022.05.24.493209"},{"key":"2023082413500502000_2023.08.21.554109v1.57","doi-asserted-by":"publisher","DOI":"10.1101\/2021.05.26.445687"},{"key":"2023082413500502000_2023.08.21.554109v1.58"},{"key":"2023082413500502000_2023.08.21.554109v1.59","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.11.6307"},{"key":"2023082413500502000_2023.08.21.554109v1.60","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.26.4.1333-1346.2006"},{"key":"2023082413500502000_2023.08.21.554109v1.61","doi-asserted-by":"publisher","DOI":"10.1038\/gt.2017.34"},{"key":"2023082413500502000_2023.08.21.554109v1.62","doi-asserted-by":"publisher","DOI":"10.1101\/gad.1941310"},{"key":"2023082413500502000_2023.08.21.554109v1.63","doi-asserted-by":"publisher","DOI":"10.1038\/nature10485"},{"key":"2023082413500502000_2023.08.21.554109v1.64","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.0050073"},{"key":"2023082413500502000_2023.08.21.554109v1.65","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2008.01.014"},{"key":"2023082413500502000_2023.08.21.554109v1.66","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkl967"},{"key":"2023082413500502000_2023.08.21.554109v1.67","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/9.2.259"},{"key":"2023082413500502000_2023.08.21.554109v1.68","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.160181697"},{"key":"2023082413500502000_2023.08.21.554109v1.69","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1002\/wrna.31","article-title":"HITS-CLIP: panoramic views of protein-RNA regulation in living cells","volume":"1","year":"2010","journal-title":"Wiley Interdiscip Rev RNA"},{"key":"2023082413500502000_2023.08.21.554109v1.70","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2018.01.005"},{"key":"2023082413500502000_2023.08.21.554109v1.71","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2077-3"},{"key":"2023082413500502000_2023.08.21.554109v1.72","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2017.016"},{"key":"2023082413500502000_2023.08.21.554109v1.73","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the false discovery rate: a practical and powerful approach to multiple testing","volume":"57","year":"1995","journal-title":"J. Roy. Statist. Soc. B"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2023.08.21.554109","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T05:06:19Z","timestamp":1768453579000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2023.08.21.554109"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,21]]},"references-count":73,"URL":"https:\/\/doi.org\/10.1101\/2023.08.21.554109","relation":{},"subject":[],"published":{"date-parts":[[2023,8,21]]},"subtype":"preprint"}}