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Updated: May 3, 2026
Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
Published on: June 6, 2020
Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual
Zuyao Ni1, Nujhat Ahmed1,2, Syed Nabeel-Shah1,2
1Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.
Abstract:
Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.
Insights
Researchers developed a new CRISPR/Cas9 screening method to identify factors involved in messenger RNA 3' end processing (cleavage and polyadenylation). This system successfully identified known factors and new potential regulators like CCNK/CDK12.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Messenger RNA (mRNA) precursors undergo 3' end processing, a crucial step for gene expression, involving cleavage and polyadenylation (CPA).
- Identifying human CPA factors has relied on biochemical and proteomic methods, lacking efficient genome-wide genetic screening approaches.
- Understanding CPA factors is vital for regulating gene expression and cellular function.
Purpose of the Study:
- To develop and validate a novel genome-wide screening method for identifying human cleavage and polyadenylation (CPA) factors.
- To apply this system to discover new CPA factors and elucidate their roles in mRNA processing.
- To provide a platform for studying CPA requirements in diverse cellular contexts.
Main Methods:
- Development of a dual fluorescence readthrough reporter system to measure 3' end processing efficiency in living cells.
- Integration of the reporter system with a human genome-wide CRISPR/Cas9 library for large-scale genetic screening.
- Analysis of screening results to identify genes modulating CPA efficiency.
Main Results:
- The screening successfully identified known core and auxiliary CPA factors, validating the system's reliability.
- New potential CPA factors, including CCNK/CDK12 and RPRD1B, were identified.
- RPRD1B was characterized as an RNA-binding factor regulating RNA polymerase II release during 3' end processing.
Conclusions:
- The dual fluorescence reporter and CRISPR/Cas9 screening system is a powerful and reliable tool for identifying human CPA factors.
- This approach expands the known repertoire of CPA factors and provides insights into novel regulatory mechanisms.
- The developed platform facilitates future investigations into the functional roles of CPA factors in various biological settings.
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