CRISPR-CBEI

CRISPR-CBEI designs single guide RNAs (sgRNAs) for Cytosine Base Editor (CBE)-mediated gene inactivation by predicting cytosine-to-thymidine edits that convert codons (CAA, CAG, CGA, TGG) into stop codons and evaluating target sites for loss-of-function studies.

Key Features:

• sgRNA design for CBE-mediated inactivation: Designs sgRNAs targeting cytosines within coding sequences to enable C-to-T conversions that generate premature stop codons.
• Codon-targeting for stop generation: Identifies cytosines within CAA, CAG, CGA, and TGG codons whose C-to-T conversion yields stop codons.
• ORF identification: Performs open reading frame (ORF) identification to locate coding regions and targetable codons.
• CBE customization: Supports customization of CBE parameters to accommodate different editors and editing windows.
• Off-target searching: Performs off-target searching against user-defined genome files of unlimited size.
• Batch processing and library design: Supports batch processing to generate gene-inactivation libraries across multiple coding DNA sequences (CDSs).
• Summarization: Produces summaries of sgRNA candidates and target-site evaluations.
• Comparative evaluation: Enables comparative evaluation of CBE variants and target genomes.

Scientific Applications:

• Loss-of-function studies: Facilitates generation of premature stop codons for targeted loss-of-function experiments.
• Recombination-deficient species: Enables gene inactivation in recombination-deficient species where donor-template repair is limited.
• Gene-inactivation libraries: Supports creation of gene-inactivation libraries across multiple CDSs for functional genomics screens.
• CBE performance evaluation: Enables comparative assessment of CBE activity and target-site suitability across genomes.
• Precise single-nucleotide editing: Targets precise C-to-T conversions at single-nucleotide resolution without inducing double-strand DNA breaks or requiring donor templates.

Methodology:

Performs ORF identification, predicts cytosine-to-thymidine conversions within target codons, conducts off-target searches against user-defined genome files, supports batch processing and CBE parameter customization, and models base editing using a catalytically inactive Cas nuclease fused to a nucleotide deaminase to achieve single-nucleotide C-to-T edits without double-strand breaks or donor templates.