gRNA-SeqRET

gRNA-SeqRET automates design and sequence extraction for pooled guide RNA (gRNA) libraries to support CRISPR-based high-throughput functional genomic screening and the construction of homology arms for insertion or deletion constructs across prokaryotic and eukaryotic genomes.

Key Features:

• Built on CCTop: Integrates CCTop for gRNA target prediction and scoring.
• Automated gRNA library design: Provides an automated and scalable workflow to design pooled gRNA libraries.
• Genomic region and whole-genome targeting: Designs guides that target user-specified genomic regions or entire genomes.
• Sequence extraction for homology arms: Extracts sequences relative to genes or other genomic features to construct homology arms for insertion or deletion constructs.
• Cross-genome applicability assessment: Evaluates whether a designed gRNA library remains applicable across closely related genomes using Average Nucleotide Identity (ANI) comparisons (e.g., >95%).
• Supports prokaryotic and eukaryotic genomes: Applies design and extraction workflows to both prokaryotic and eukaryotic organisms.
• Pooled gRNA library construction: Enables assembly of pooled guide RNA libraries for high-throughput experiments.
• Facilitates gene–phenotype and sequence–function analyses: Supports association of genes with phenotypes and elucidation of sequence–function relationships via designed libraries.

Scientific Applications:

• CRISPR-based genetic screens: Generation of pooled gRNA libraries for high-throughput loss- or gain-of-function screens.
• Functional genomics: Targeted interrogation of gene function and sequence–function relationships across diverse organisms.
• Comparative genomics: Assessment of library transferability and applicability across closely related strains or species using ANI thresholds.
• Genome engineering: Design of homology arms and guides for insertion or deletion construct development.

Methodology:

Implements CCTop and customized Python scripts in an automated, scalable workflow to design gRNA libraries, extract sequences relative to genes/genomic features for homology arms, and evaluate library applicability across genomes via ANI (>95%) comparisons.