Abstract
Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR–Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3–4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.
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Data availability
The data used to generate the example results shown in Fig. 5 were originally published in ref. 23. All sequencing datasets have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GSE149690, GSE149691 and GSE149692) and National Omics Data Encyclopedia (OEP000887, OEP000888 and OEP000889). All the plasmids, cell lines and reagents presented in the paper are available on request from the corresponding author.
Code availability
The custom Perl and Shell scripts for the computational pipeline of the CDCscreen in this paper are available at https://github.com/YangLab/CDCscreen.
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Acknowledgements
We thank W. Xue for providing sequencing data-analysis support. This study was supported by the National Key R&D Program of China (2021YFA1300501), the National Natural Science Foundation of China (NSFC) (31725009, 91940303 and 31821004), the Center for Excellence in Molecular Cell Science (CEMCS) (2020DF03) and the HHMI International Program (55008728) to L.-L.C.; and NSFC (32101042) to S.L. L.-L.C. acknowledges support from the Xplorer Prize.
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L.-L.C. supervised and conceived the project. L.-L.C., S.L. and H.W. designed the experiments. S.L. and H.W. performed the experiments. L.-L.C., S.L. and H.W. wrote the manuscript.
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Nature Protocols thanks David Gilot and Jun Wei Pek for their contribution to the peer review of this work.
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Li, S. et al. Nat. Methods 18, 51–59 (2021): https://doi.org/10.1038/s41592-020-01011-4
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Li, S., Wu, H. & Chen, LL. Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system. Nat Protoc 17, 2085–2107 (2022). https://doi.org/10.1038/s41596-022-00715-5
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DOI: https://doi.org/10.1038/s41596-022-00715-5
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