CasCADE: Cas-CRISPR Automated Design and Evaluation for targeted gRNA detection assays

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• CRISPR technologies offer enhanced specificity and sensitivity for molecular detection compared to PCR.
• Effective CRISPR-based detection relies on precise guide RNA (gRNA) design, which is often complex and time-consuming.
• Current methods for gRNA design can be computationally intensive, hindering rapid application in biosurveillance.

Purpose of the Study:

• To introduce Cas-CRISPR Automated Design and Evaluation (CasCADE), a novel software pipeline for *in silico* design of CRISPR-based detection assays.
• To demonstrate the efficiency and accuracy of CasCADE in designing gRNA sequences for diverse target organisms.
• To provide a tool that accelerates the development of CRISPR detection assays for enhanced biosurveillance and pandemic preparedness.

Main Methods:

• CasCADE utilizes k-mer set operations for efficient processing of large datasets, outperforming traditional multiple sequence alignment.
• The software employs an agnostic whole-genome approach to maximize the discovery of potential gRNA targets.
• CasCADE offers flexibility for users to perform design, candidate evaluation, or both, based on specific needs.

Main Results:

• CasCADE successfully designed 15 gRNA sequences across nine different organisms, demonstrating its broad applicability.
• The software provides a rapid time to answer for gRNA design, crucial for timely biosurveillance.
• Laboratory validation confirmed the viability of the *in silico* designs generated by CasCADE.

Conclusions:

• CasCADE is a scalable and modular software platform that significantly streamlines the design of CRISPR-based molecular detection assays.
• The rapid and accurate design capabilities of CasCADE enhance the potential of CRISPR technology for pathogen detection and biosurveillance.
• This tool represents a timely contribution to pandemic preparedness by enabling faster development of sensitive and specific diagnostic tools.