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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
1Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC, 27401, USA.
Small (Weinheim an Der Bergstrasse, Germany)
|June 6, 2024
Summary
Compartmentalized CRISPR reactions (CCR) enable rapid in vitro screening of guide RNA (gRNA) variants for CRISPR-Cas9 gene editing. This method efficiently validates gRNA activity and specificity, streamlining research applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR ribonucleoproteins (RNPs) utilize guide RNA (gRNA) spacers to direct nuclease activity for targeted DNA mutation.
- Predicting gRNA spacer sequence-dependent nuclease activity and off-target effects remains challenging.
- In vitro validation of individual gRNAs is a necessary but time-consuming step in CRISPR applications.
Purpose of the Study:
- To develop a method for simultaneous in vitro screening of numerous CRISPR effector activity and specificity combinations.
- To validate computational predictions of gRNA performance.
- To streamline the generation and validation of gRNAs for biological and biomedical research.
Main Methods:
- Development of compartmentalized CRISPR reactions (CCR) using water-in-oil microemulsions.
- Confinement of the complete CRISPR reaction (gRNA transcription, RNP formation, target cleavage) within individual microemulsions.
- Screening of large numbers of spacer/target/off-target combinations in parallel.
Main Results:
- CCR enables simultaneous screening of CRISPR effector activity and specificity in vitro.
- Demonstrated screening of hundreds of thousands of extended gRNA (x-gRNA) variants.
- Identified x-gRNAs that maintain high on-target activity while blocking off-target cleavage.
Conclusions:
- Compartmentalized CRISPR reactions (CCR) provide an efficient platform for high-throughput gRNA validation.
- CCR significantly accelerates the gRNA generation and validation pipeline.
- This approach is expected to benefit diverse applications in biological and biomedical research.
