GuideFinder

GuideFinder designs CRISPR/Cas9 guide RNAs across bacterial genomes to enable targeted CRISPRi and CRISPRa for systematic interrogation of gene function.

Key Features:

• Genome-wide Guide Design: Designs guides across any annotated bacterial genome by scanning NGG protospacer-adjacent motif (PAM) sites from an input annotated genome and corresponding FASTA file.
• Customizable Parameters: Applies user-defined design parameters to accommodate genetic diversity and draft genome annotations.
• Off-Target Filtering: Filters candidate guides for off-target matches using adjustable thresholds and supports iterative parameter relaxation to recover guides for challenging targets.
• Paired Guide Identification: Identifies paired guide sequences for multiplexed targeting, a capability validated experimentally.
• High Success Rate: Has identified guides for approximately 95% of genes across tested bacterial genomes.
• Functional Validation: Guides have been functionally validated in CRISPRi essential gene knockdown experiments in two staphylococcal species.

Scientific Applications:

• Systematic Gene-Function Interrogation: Enables CRISPRi and CRISPRa experiments to assess gene function across bacterial genomes.
• Diverse and Draft Genome Targeting: Supports guide design in diverse bacterial species and draft genome assemblies for comparative and functional genomics.
• Multiplexed Targeting: Facilitates paired-guide or multiplexed targeting strategies for complex genetic perturbations.
• Applied Microbial Manipulation: Supports studies aimed at microbial manipulation, control, and validation of essential genes.

Methodology:

Analyzes NGG PAM sites in an input annotated bacterial genome and corresponding FASTA, applies user-defined filters including off-target criteria, iteratively adjusts stringency thresholds, and selects individual or paired guides.