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Compartmentalized CRISPR Reactions (CCR) for High-Throughput Screening of Guide RNA Potency and Specificity
Tinku Supakar1, Ashley Herring-Nicholas1, Eric A Josephs1
1T. Supakar, A. H. Nicholas, E. A. Josephs Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro Greensboro, NC, USA 27401.
Biorxiv : the Preprint Server for Biology
|May 20, 2024
Summary
Compartmentalized CRISPR reactions (CCR) enable rapid in vitro screening of guide RNA (gRNA) activity and specificity. This method efficiently validates numerous gRNA variants, streamlining CRISPR research and development.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR ribonucleoproteins (RNPs) rely on guide RNA (gRNA) spacers for targeted DNA cleavage, but activity varies unpredictably with gRNA sequence.
- Predictive computational tools aid in assessing CRISPR effector activity and off-target effects, yet in vitro validation of individual gRNAs remains essential.
Approach:
- Developed compartmentalized CRISPR reactions (CCR) using water-in-oil microemulsions to encapsulate and screen gRNA transcription, RNP formation, and target cleavage simultaneously.
- CCR allows for high-throughput, parallel validation of numerous gRNA candidates designed by computational tools.
- Demonstrated CCR's capability to screen hundreds of thousands of extended gRNA (x-gRNA) variants for enhanced specificity and on-target activity.
Key Points:
- CCR integrates multiple CRISPR workflow steps within microemulsions for simultaneous in vitro screening.
- Enables rapid validation of large gRNA libraries, including extended gRNA variants.
- Successfully screened x-gRNAs to block off-target cleavage while preserving on-target activity.
Conclusions:
- CCR significantly streamlines the generation and validation of gRNAs for CRISPR applications.
- This platform accelerates the development of precise gene editing tools for biological and biomedical research.
- CCR facilitates the optimization of gRNA design for improved CRISPR system performance and safety.
