{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T15:53:39Z","timestamp":1774713219664,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,25]]},"group-title":"Synthetic Biology","reference-count":79,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T00:00:00Z","timestamp":1774396800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,25]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  In gene editing, CRISPR\/Cas approaches are often limited by off-target effects. In in vivo approaches involving multiple cell types, off-targets may result from unintended targeting of the wrong cells. In this work, we propose a solution to this limitation by using a transcribed intron of the target gene as an endogenous trigger (intron triggers) for a novel conditional guide RNA (intcgRNA). In vitro, intcgRNAs were responsive to the presence of the trigger. As a proof-of-concept, the human IL2 receptor subunit gamma gene (\n                  <jats:italic>IL2RG<\/jats:italic>\n                  ) was then targeted using both the intcgRNA and the corresponding conventional crRNA in two cell lines: the lymphocytic HPB-ALL cell line, where IL2RG is highly expressed, and the epithelial HeLa cell line, where it is not. Sanger sequencing revealed that the crRNA and intcgRNA Cas9 complexes edited\n                  <jats:italic>IL2RG<\/jats:italic>\n                  with similar efficiency in HPB-ALL, whereas only the crRNA edited\n                  <jats:italic>IL2RG<\/jats:italic>\n                  in HeLa. This shows that intcgRNA avoids targeting unwanted cells that do not express the target gene, which is particularly relevant for in vivo targeting. The triggers of choice for conditional guides have been microRNAs, but as short intronic RNAs are far more diverse, trigger introns could become biomarkers of cell identity that improve the precision of CRISPR-based manipulations in vivo.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>GRAPHICAL ABSTRACT<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"714022v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.24.714022","type":"posted-content","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T04:26:36Z","timestamp":1774499196000},"source":"Crossref","is-referenced-by-count":0,"title":["Endogenous intronic RNA tightly controls Cas9\/CRISPR-mediated gene editing in human cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-1613-8227","authenticated-orcid":false,"given":"Andr\u00e9 Lemos","family":"Carneiro","sequence":"first","affiliation":[{"name":"Life Sciences Department, NOVA School of Science and Technology, Portugal"},{"name":"INESC-ID, Institute for Systems and Computer Engineering, Research and Development, Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9861-8241","authenticated-orcid":false,"given":"Jo\u00e3o","family":"Proen\u00e7a","sequence":"additional","affiliation":[{"name":"Gulbenkian Institute for Molecular Medicine, Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5272-0016","authenticated-orcid":false,"given":"Ehsan","family":"Valiollahi","sequence":"additional","affiliation":[{"name":"Plant Cell Biotechnology Lab, Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa, Oeiras, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0010-8607","authenticated-orcid":false,"given":"Vasco M.","family":"Barreto","sequence":"additional","affiliation":[{"name":"Life Sciences Department, NOVA School of Science and Technology, Portugal"},{"name":"Egas Moniz Centre for Interdisciplinary Research (CiiEM), Egas Moniz School of Health & Science, Portugal"},{"name":"Research Unit on Applied Molecular Biosciences (UCIBIO) and Associate Laboratory Institute for Health and Bioeconomy (i4HB), Portugal"}]}],"member":"54368","reference":[{"key":"2026032808200872000_2026.03.24.714022v1.1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"2026032808200872000_2026.03.24.714022v1.2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1208507109"},{"key":"2026032808200872000_2026.03.24.714022v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.1231143"},{"key":"2026032808200872000_2026.03.24.714022v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.1232033"},{"key":"2026032808200872000_2026.03.24.714022v1.5","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1182\/blood-2024-211182","article-title":"SAGES1: Clinical Translation of CRISPR Genome Editing Strategy to Induce Fetal Hemoglobin to Treat Sickle Cell Disease","volume":"144","year":"2024","journal-title":"Blood"},{"key":"2026032808200872000_2026.03.24.714022v1.6","doi-asserted-by":"publisher","DOI":"10.1101\/gr.171322.113"},{"key":"2026032808200872000_2026.03.24.714022v1.7","doi-asserted-by":"crossref","first-page":"6261","DOI":"10.3390\/ijms24076261","article-title":"Current Bioinformatics Tools to Optimize CRISPR\/Cas9 Experiments to Reduce Off-Target Effects","volume":"24","year":"2023","journal-title":"Int. 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