Large scale screening of CRISPR guide RNAs using an optimized high throughput robotics system.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR/Cas systems rely on CRISPR guide RNAs (crRNAs) for targeting specific genetic sequences.
• crRNA design is complex, influenced by Cas protein type, target, and environmental factors.
• Optimizing crRNA design is crucial for enhancing CRISPR/Cas system efficacy, particularly for Cas13a RNA targeting.

Purpose of the Study:

• To develop and validate a high-throughput method for screening large numbers of candidate crRNAs for Cas13a activation.
• To identify key factors influencing crRNA efficacy in Cas13a-mediated collateral RNA cleavage.
• To establish a generalizable method for optimizing crRNA designs for Cas12 and Cas13 systems.

Main Methods:

• A high-throughput screening platform was established to test hundreds of candidate crRNAs against a model target RNA (Y. pestis lcrV gene).
• crRNAs targeting tiled regions of the gene transcript were synthesized and screened for their ability to activate Cas13a collateral activity.
• Factors evaluated included crRNA/target sequence, size, secondary structures, and the commercial source of DNA oligomers used for crRNA synthesis.

Main Results:

• Initial screening identified 271 out of 296 (91.5%) tested guide RNAs capable of detecting the target RNA.
• Protocol optimization improved detection rates to 287 out of 296 (97%) guide RNAs.
• crRNA efficacy was found to be significantly dependent on the source of DNA oligomers, rather than target region or crRNA physical properties.

Conclusions:

• The developed high-throughput method enables efficient screening and optimization of crRNAs for Cas13a collateral RNA cleavage.
• The source of DNA oligomers used for crRNA synthesis is a critical determinant of crRNA efficacy.
• This screening approach has broad applicability for optimizing guide RNA designs in various CRISPR/Cas systems, including Cas12 and Cas13.