{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T12:17:07Z","timestamp":1787314627194,"version":"build-2736575974"},"posted":{"date-parts":[[2024,2,1]]},"group-title":"Molecular Biology","reference-count":50,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,2,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>RNA editing offers the opportunity to introduce either stable or transient modifications to nucleic acid sequence without permanent off-target effects, but installation of arbitrary edits into the transcriptome is currently infeasible. Here, we describe Programmable RNA Editing &amp; Cleavage for Insertion, Substitution, and Erasure (PRECISE), a versatile RNA editing method for writing RNA of arbitrary length and sequence into existing pre-mRNAs via 5\u2032 or 3\u2032 trans-splicing. In trans-splicing, an exogenous template is introduced to compete with the endogenous pre-mRNA, allowing for replacement of upstream or downstream exon sequence. Using Cas7-11 cleavage of pre-mRNAs to bias towards editing outcomes, we boost the efficiency of RNA trans-splicing by 10\u2013100 fold, achieving editing rates between 5\u201350% and 85% on endogenous and reporter transcripts, respectively, while maintaining high-fidelity. We demonstrate PRECISE editing across 11 distinct endogenous transcripts of widely varying expression levels, showcasing more than 50 types of edits, including all 12 possible transversions and transitions, insertions ranging from 1 to 1,863 nucleotides, and deletions. We show high efficiency replacement of exon 4 of MECP2, addressing most mutations that drive the Rett Syndrome; editing of SHANK3 transcripts, a gene involved in Autism; and replacement of exon 1 of HTT, removing the hallmark repeat expansions of Huntington\u2032s disease. Whole transcriptome sequencing reveals the high precision of PRECISE editing and lack of off-target trans-splicing activity. Furthermore, we combine payload engineering and ribozymes for protein-free, high-efficiency trans-splicing, with demonstrated efficiency in editing HTT exon 1 via AAV delivery. We show that the high activity of PRECISE editing enables editing in non-dividing neurons and patient-derived Huntington\u2019s disease fibroblasts. PRECISE editing markedly broadens the scope of genetic editing, is straightforward to deliver over existing gene editing tools like prime editing, lacks permanent off-targets, and can enable any type of genetic edit large or small, including edits not otherwise possible with existing RNA base editors, widening the spectrum of addressable diseases.<\/jats:p>","DOI":"10.1101\/2024.01.31.578223","type":"posted-content","created":{"date-parts":[[2024,2,2]],"date-time":"2024-02-02T00:20:10Z","timestamp":1706833210000},"source":"Crossref","is-referenced-by-count":15,"title":["Programmable RNA writing with trans-splicing"],"prefix":"10.64898","author":[{"given":"Cian","family":"Schmitt-Ulms","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alisan","family":"Kayabolen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcos","family":"Manero-Carranza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keira","family":"Donnelly","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabrina Pia","family":"Nuccio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuki","family":"Kato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Nishimasu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2757-2175","authenticated-orcid":false,"given":"Jonathan S.","family":"Gootenberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7979-3220","authenticated-orcid":false,"given":"Omar O.","family":"Abudayyeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2024020505350871000_2024.01.31.578223v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nature17946"},{"key":"2024020505350871000_2024.01.31.578223v1.2","doi-asserted-by":"crossref","unstructured":"Nishida, K. et al. 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