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Identification of Circular RNAs using RNA Sequencing
Published on: November 14, 2019
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Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system
Siqi Li1, Hao Wu1, Ling-Ling Chen2,3,4
1State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
Nature Protocols
|July 13, 2022
Summary
This study introduces a CRISPR-Cas13 based protocol for large-scale screening of circular RNAs (circRNAs) in human cells. The method efficiently identifies circRNAs impacting cell growth without affecting messenger RNAs (mRNAs).
Area of Science:
- Molecular Biology
- Genetics
- RNA Biology
Background:
- Circular RNAs (circRNAs) are key regulators of gene expression.
- Distinguishing circRNA function from messenger RNA (mRNA) function is challenging due to sequence overlap.
- The CRISPR-Cas13 system offers a potential solution for specific circRNA targeting.
Purpose of the Study:
- To present a detailed protocol for large-scale functional circRNA screening using the RfxCas13d/BSJ-gRNA system.
- To enable the identification of circRNAs that influence cell growth.
- To provide a method that specifically targets circRNAs without affecting their cognate mRNAs.
Main Methods:
- Utilizing a programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system with guide RNAs (gRNAs) targeting back-splicing junction (BSJ) sites.
- Designing and constructing a gRNA library for efficient transduction.
- Analyzing screening results and validating functional circRNA candidates in human cell lines.
Main Results:
- The RfxCas13d/BSJ-gRNA system effectively and specifically discriminates circRNAs from mRNAs.
- The protocol facilitates large-scale functional screening of circRNAs.
- The method can identify circRNAs affecting cell growth under various conditions.
Conclusions:
- The RfxCas13d/BSJ-gRNA protocol provides a robust method for circRNA functional screening.
- This approach allows for the specific investigation of circRNA roles in cellular processes.
- The protocol is applicable in both cellular and in vivo models for identifying functionally significant circRNAs.
