Efficient, specific, and combinatorial control of endogenous exon splicing with dCasRx-RBM25
Open accessAbstract
Efficient targeted control of splicing is a major goal of functional genomics and therapeutic applications. Guide (g)RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility of these systems. However, efficient, specific, and generalizable activation of endogenous exons using this approach has not been previously reported. By screening over 300 dCasRx-splicing factor fusion proteins tethered to splicing reporters, we identify dCasRx-RBM25 as a potent activator of exons. Moreover, dCasRx-RBM25 efficiently activates the splicing of ∼90% of targeted endogenous alternative exons and displays high on-target specificity. Using gRNA arrays for combinatorial targeting, we demonstrate that dCasRx-RBM25 enables multiplexed activation and repression of exons. Using this feature, the targeting of neural-regulated exons in Ptpb1 and Puf60 in embryonic stem cells reveals combinatorial effects on downstream alternative splicing events controlled by these factors. Collectively, our results enable versatile, combinatorial exon-resolution functional assays and splicing-directed therapeutic applications.
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Screening of 453 splicing factors showed RBM25 activates splicing in >90% of targeted exons across contexts, including MAPT exon 10.
“Moreover, screening of 453 splicing factors showed RBM25 activates splicing in >90% of targeted exons across contexts, including MAPT exon 1057.”
From The R136S mutation in the APOE3 gene confers resilience against tau pathology via inhibition of the cGAS-STING-IFN pathway (2025) · cites this paper as [57]
The Discussion reports screening 341 dCasRx-fusion proteins, not 453, and says dCasRx-RBM25 promoted inclusion of approximately 90% of tested endogenous exons. The Results report activation of MAPT exon 10. Thus the claimed screening count is incorrect, while the exon-activation finding is broadly supported. (LLM, conf=0.93)
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Research works printed but not linked: 52
- [1]1. Pan, Q. ∙ Shai, O. ∙ Lee, L.J. ... Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing Nat. Genet. 2008; 40:1413-1415 Crossref Scopus (2843) PubMed Google Scholar
- [2]2. Wang, E.T. ∙ Sandberg, R. ∙ Luo, S. ... Alternative isoform regulation in human tissue transcriptomes Nature. 2008; 456:470-476 Crossref Scopus (3903) PubMed Google Scholar
- [3]3. Sinitcyn, P. ∙ Richards, A.L. ∙ Weatheritt, R.J. ... Global detection of human variants and isoforms by deep proteome sequencing Nat. Biotechnol. 2023; 41:1776-1786 Crossref Scopus (44) PubMed Google Scholar
- [4]4. Baralle, F.E. ∙ Giudice, J. Alternative splicing as a regulator of development and tissue identity Nat. Rev. Mol. Cell Biol. 2017; 18:437-451 Crossref Scopus (802) PubMed Google Scholar
- [5]5. Ule, J. ∙ Blencowe, B.J. Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution Mol. Cell. 2019; 76:329-345 Full Text Full Text (PDF) Scopus (392) PubMed Google Scholar
- [6]6. Rogalska, M.E. ∙ Vivori, C. ∙ Valcárcel, J. Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects Nat. Rev. Genet. 2023; 24:251-269 Crossref Scopus (50) PubMed Google Scholar
- [7]7. Marasco, L.E. ∙ Kornblihtt, A.R. The physiology of alternative splicing Nat. Rev. Mol. Cell Biol. 2023; 24:242-254 Crossref Scopus (112) PubMed Google Scholar
- [8]8. Scotti, M.M. ∙ Swanson, M.S. RNA mis-splicing in disease Nat. Rev. Genet. 2016; 17:19-32 Crossref Scopus (752) PubMed Google Scholar
- [9]9. Lord, J. ∙ Baralle, D. Splicing in the Diagnosis of Rare Disease: Advances and Challenges Front. Genet. 2021; 12, 689892 Crossref Scopus (38) PubMed Google Scholar
- [10]10. Sergeeva, O.V. ∙ Shcherbinina, E.Y. ∙ Shomron, N. ... Modulation of RNA Splicing by Oligonucleotides: Mechanisms of Action and Therapeutic Implications Nucleic Acid Ther. 2022; 32:123-138 Crossref Scopus (0) PubMed Google Scholar
- [11]11. Rigo, F. ∙ Hua, Y. ∙ Krainer, A.R. ... Antisense-based therapy for the treatment of spinal muscular atrophy J. Cell Biol. 2012; 199:21-25 Crossref Scopus (0) PubMed Google Scholar
- [12]12. Gonatopoulos-Pournatzis, T. ∙ Aregger, M. ∙ Brown, K.R. ... Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform Nat. Biotechnol. 2020; 38:638-648 Crossref Scopus (0) PubMed Google Scholar
- [13]13. Thomas, J.D. ∙ Polaski, J.T. ∙ Feng, Q. ... RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons Nat. Genet. 2020; 52:84-94 Crossref Scopus (52) PubMed Google Scholar
- [14]14. Xiao, M.S., Damodaran, A.P., Kumari, B., Dickson, E., Xing, K., On, T., Parab, N., King, H.E., Perez, A.R., Guiblet, W.M., et al. (2024). Genome-scale exon perturbation screens uncover exons critical for cell fitness. Mol Cell 84, 2553–2572. https://doi.org/10.1016/j.molcel.2024.05.024. Google Scholar · DOI
- [15]15. Wang, Y. ∙ Cheong, C.G. ∙ Hall, T.M.T. ... Engineering splicing factors with designed specificities Nat. Methods. 2009; 6:825-830 Crossref Scopus (0) PubMed Google Scholar
- [16]16. Zhao, Y.Y. ∙ Mao, M.W. ∙ Zhang, W.J. ... Expanding RNA binding specificity and affinity of engineered PUF domains Nucleic Acids Res. 2018; 46:4771-4782 Crossref Scopus (0) PubMed Google Scholar
- [17]17. Fernandez Alanis, E. ∙ Pinotti, M. ∙ Dal Mas, A. ... An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects Hum. Mol. Genet. 2012; 21:2389-2398 Crossref Scopus (0) PubMed Google Scholar
- [19]19. Schneider-Poetsch, T. ∙ Chhipi-Shrestha, J.K. ∙ Yoshida, M. Splicing modulators: on the way from nature to clinic J. Antibiot. (Tokyo). 2021; 74:603-616 Crossref Scopus (0) PubMed Google Scholar
- [20]20. van Beljouw, S.P.B. ∙ Sanders, J. ∙ Rodríguez-Molina, A. ... RNA-targeting CRISPR-Cas systems Nat. Rev. Microbiol. 2023; 21:21-34 Crossref Scopus (0) PubMed Google Scholar
- [21]21. Konermann, S. ∙ Lotfy, P. ∙ Brideau, N.J. ... Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors Cell. 2018; 173:665-676.e14 Full Text Full Text (PDF) Scopus (710) PubMed Google Scholar
- [22]22. Du, M. ∙ Jillette, N. ∙ Zhu, J.J. ... CRISPR artificial splicing factors Nat. Commun. 2020; 11:2973 Crossref Scopus (64) PubMed Google Scholar
- [23]23. Núñez-Álvarez, Y. ∙ Espie-Caullet, T. ∙ Luco, R.F. A CRISPR-dCas13 RNA-editing tool to study alternative splicing Preprint at bioRxiv. 2022; Crossref Scopus (0) Google Scholar
- [24]24. Leclair, N.K. ∙ Brugiolo, M. ∙ Urbanski, L. ... Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesiss Mol. Cell. 2020; 80:648-665.e9 Full Text Full Text (PDF) Scopus (0) PubMed Google Scholar
- [25]25. Recinos, Y. ∙ Ustianenko, D. ∙ Yeh, Y.T. ... CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements Nat. Commun. 2024; 15:3839 Crossref Scopus (0) PubMed Google Scholar
- [27]27. Gonatopoulos-Pournatzis, T. ∙ Wu, M. ∙ Braunschweig, U. ... Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons Mol. Cell. 2018; 72:510-524.e12 Full Text Full Text (PDF) Scopus (0) PubMed Google Scholar
- [28]28. Lorson, C.L. ∙ Hahnen, E. ∙ Androphy, E.J. ... A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy Proc. Natl. Acad. Sci. USA. 1999; 96:6307-6311 Crossref Scopus (1263) PubMed Google Scholar
- [30]30. Agafonov, D.E. ∙ Deckert, J. ∙ Wolf, E. ... Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method Mol. Cell. Biol. 2011; 31:2667-2682 Crossref Scopus (151) PubMed Google Scholar
- [31]31. Kastner, B. ∙ Will, C.L. ∙ Stark, H. ... Structural Insights into Nuclear pre-mRNA Splicing in Higher Eukaryotes Cold Spring Harb. Perspect. Biol. 2019; 11, a032417 Crossref Scopus (81) PubMed Google Scholar
- [34]34. Gottschalk, A. ∙ Tang, J. ∙ Puig, O. ... A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins RNA. 1998; 4:374-393 PubMed Google Scholar
- [35]35. Li, X. ∙ Liu, S. ∙ Jiang, J. ... CryoEM structure of Saccharomyces cerevisiae U1 snRNP offers insight into alternative splicing Nat. Commun. 2017; 8:1035 Crossref Scopus (0) PubMed Google Scholar
- [36]36. Fortes, P. ∙ Longman, D. ∙ McCracken, S. ... Identification and characterization of RED120: a conserved PWI domain protein with links to splicing and 3′-end formation FEBS Lett. 2007; 581:3087-3097 Crossref Scopus (0) PubMed Google Scholar
- [37]37. Carlson, S.M. ∙ Soulette, C.M. ∙ Yang, Z. ... RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation J. Biol. Chem. 2017; 292:13381-13390 Full Text Full Text (PDF) Scopus (0) PubMed Google Scholar
- [38]38. Wessels, H.H. ∙ Méndez-Mancilla, A. ∙ Guo, X. ... Massively parallel Cas13 screens reveal principles for guide RNA design Nat. Biotechnol. 2020; 38:722-727 Crossref Scopus (200) PubMed Google Scholar
- [39]39. Guo, X. ∙ Rahman, J.A. ∙ Wessels, H.H. ... Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens Cell Genom. 2021; 1, 100001 Full Text Full Text (PDF) Scopus (0) PubMed Google Scholar
- [40]40. Liu, F. ∙ Gong, C.X. Tau exon 10 alternative splicing and tauopathies Mol. Neurodegener. 2008; 3:8 Crossref Scopus (0) PubMed Google Scholar
- [41]41. Parras, A. ∙ Anta, H. ∙ Santos-Galindo, M. ... Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing Nature. 2018; 560:441-446 Crossref Scopus (93) PubMed Google Scholar
- [42]42. Liu, Z. ∙ Jillette, N. ∙ Robson, P. ... Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold Nucleic Acids Res. 2023; 51, e77 Crossref Scopus (0) Google Scholar
- [44]44. Gong, D. ∙ Yang, F. ∙ Li, F. ... Crystal structure and functional characterization of the human RBM25 PWI domain and its flanking basic region Biochem. J. 2013; 450:85-94 Crossref Scopus (0) PubMed Google Scholar
- [45]45. Zhou, A. ∙ Ou, A.C. ∙ Cho, A. ... Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5′ splice site selection Mol. Cell. Biol. 2008; 28:5924-5936 Crossref Scopus (0) PubMed Google Scholar
- [46]46. Le Sénéchal, R. ∙ Keruzoré, M. ∙ Quillévéré, A. ... Alternative splicing of BCL-x is controlled by RBM25 binding to a G-quadruplex in BCL-x pre-mRNA Nucleic Acids Res. 2023; 51:11239-11257 Crossref Scopus (0) PubMed Google Scholar
- [47]47. Boutz, P.L. ∙ Stoilov, P. ∙ Li, Q. ... A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons Genes Dev. 2007; 21:1636-1652 Crossref Scopus (429) PubMed Google Scholar
- [49]49. Han, H. ∙ Best, A.J. ∙ Braunschweig, U. ... Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis Mol. Cell. 2022; 82:2982-2999.e14 Full Text Full Text (PDF) Scopus (9) PubMed Google Scholar
- [50]50. Roth, J.F. ∙ Braunschweig, U. ∙ Wu, M. ... Systematic analysis of alternative exon-dependent interactome remodeling reveals multitasking functions of gene regulatory factors Mol. Cell. 2023; 83:4222-4238.e10 Full Text Full Text (PDF) Scopus (0) PubMed Google Scholar
- [52]52. Gapinske, M. ∙ Luu, A. ∙ Winter, J. ... CRISPR-SKIP: programmable gene splicing with single base editors Genome Biol. 2018; 19:107 Crossref Scopus (127) PubMed Google Scholar
- [53]53. Zhang, C. ∙ Konermann, S. ∙ Brideau, N.J. ... Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d Cell. 2018; 175:212-223.e17 Full Text Full Text (PDF) Scopus (155) PubMed Google Scholar
- [54]54. Raguram, A. ∙ Banskota, S. ∙ Liu, D.R. Therapeutic in vivo delivery of gene editing agents Cell. 2022; 185:2806-2827 Full Text Full Text (PDF) Scopus (153) PubMed Google Scholar
- [55]55. Tapial, J. ∙ Ha, K.C.H. ∙ Sterne-Weiler, T. ... An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms Genome Res. 2017; 27:1759-1768 Crossref Scopus (237) PubMed Google Scholar
- [56]56. Zhao, S. ∙ Jiang, E. ∙ Chen, S. ... PiggyBac transposon vectors: the tools of the human gene encoding Transl. Lung Cancer Res. 2016; 5:120-125 Crossref Scopus (0) PubMed Google Scholar
- [57]57. Schertzer, M.D. ∙ Thulson, E. ∙ Braceros, K.C.A. ... A piggyBac-based toolkit for inducible genome editing in mammalian cells RNA. 2019; 25:1047-1058 Crossref Scopus (24) PubMed Google Scholar
- [58]58. Dobin, A. ∙ Davis, C.A. ∙ Schlesinger, F. ... STAR: ultrafast universal RNA-seq aligner Bioinformatics. 2013; 29:15-21 Crossref Scopus (26617) PubMed Google Scholar
- [59]59. McCarthy, D.J. ∙ Chen, Y. ∙ Smyth, G.K. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation Nucleic Acids Res. 2012; 40:4288-4297 Crossref Scopus (3367) PubMed Google Scholar
- [60]60. Wessels, H.H. ∙ Stirn, A. ∙ Méndez-Mancilla, A. ... Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning Nat. Biotechnol. 2024; 42:628-637 Crossref Scopus (0) PubMed Google Scholar
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Naguib, S., Lopez-Lee, C., Torres, E. R., Lee, S.-I., Zhu, J., Zhu, D., Ye, P., Norman, K., Zhao, M., Wong, M. Y., et al. (2025) · Immunity · doi:10.1016/j.immuni.2025.05.023 · Cited by 32 · see citing works
- Authors
- Li, J. D., Taipale, M., & Blencowe, B. J.
- Affiliations
- University of Toronto (CA)
- Corresponding author
- Taipale, Mikko
- Blencowe, Benjamin J.
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2024-07
- Volume
- 84
- Issue
- 13
- Pages
- 2573-2589.e5
- ISSN
- 1097-2765 (ISSN-L)
- OpenAlex
- W4399970126
- Cited by
- 31
- References
- 4
- Citations by year
- 2026: 12 · 2025: 8 · 2024: 11
- FWCI
- 4.01
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 375 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Molecular Cell (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Canada Research Chairs (LU506463)
- Canadian Institutes of Health Research
- Simons Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- alternative splicing, PUF60, embryonic stem cells
- MeSH terms
- RNA Splicing Factors, RNA, Guide, CRISPR-Cas Systems, Animals, Exons, Humans, RNA-Binding Proteins, Alternative Splicing, Mice, HEK293 Cells, CRISPR-Cas Systems
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