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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
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Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing
Joseph M Replogle1,2,3,4,5, Thomas M Norman3,4,5,6, Albert Xu1,3,4,5
1Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
Nature Biotechnology
|April 2, 2020
Summary
Direct-capture Perturb-seq revolutionizes single-cell CRISPR screens by directly sequencing guide RNAs with transcriptomes. This enables high-throughput genetic interaction studies and improves CRISPR tool efficacy for mammalian gene function exploration.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Single-cell CRISPR screens are powerful for studying gene function but limited by indirect single-guide RNA (sgRNA) detection.
- Existing methods require indirect indexing, hindering scalability and combinatorial screening.
Purpose of the Study:
- To introduce direct-capture Perturb-seq, a method for direct sequencing of sgRNAs alongside single-cell transcriptomes.
- To enable high-throughput pooled and combinatorial CRISPR screens.
- To enhance the efficacy of CRISPR interference and activation in single-cell applications.
Main Methods:
- Direct-capture Perturb-seq: sequencing expressed sgRNAs with single-cell transcriptomes.
- Application in pooled and combinatorial CRISPR screens, including dual-guide vectors.
- Investigation of genetic interactions, specifically epistatic interactions between cholesterol biogenesis and DNA repair.
- Utilizing multiple sgRNAs per gene for enhanced CRISPR interference/activation.
- Employing hybridization-based target enrichment for sensitive transcript sequencing.
Main Results:
- Direct-capture Perturb-seq enables detection of multiple sgRNAs per cell, facilitating combinatorial screening.
- Successfully dissected epistatic interactions between cholesterol biogenesis and DNA repair pathways.
- Demonstrated improved CRISPR interference and activation efficacy using multiple sgRNAs per gene.
- Showcased sensitive and specific sequencing of informative transcripts via target enrichment.
Conclusions:
- Direct-capture Perturb-seq significantly advances single-cell CRISPR screening capabilities.
- The method supports high-throughput genetic interaction studies and combinatorial perturbations.
- It enhances CRISPR tool performance, enabling more efficient exploration of gene function and regulatory networks.
