indCAPS

indCAPS designs derived cleaved amplified polymorphic sequence (dCAPS) primers to detect small insertions and deletions (indels) from CRISPR/Cas9 mutagenesis by creating or modifying restriction enzyme recognition sites in PCR amplicons.

Key Features:

• dCAPS primer design: Designs primers that incorporate intentional mismatches to create or modify restriction enzyme recognition sites for allele discrimination.
• Indel accommodation: Handles sequences containing small insertions and deletions (indels) that are problematic for conventional dCAPS design tools.
• CRISPR/Cas9 focus: Targets detection of CRISPR/Cas9-induced editing events, including subtle indels.
• Implementation: Implemented in Python and developed to be species-agnostic for use across taxa.
• CAPS compatibility: Produces primer pairs compatible with CAPS/dCAPS assays involving PCR amplification and restriction enzyme digestion.

Scientific Applications:

• CRISPR/Cas9 screening: Enables screening and genotyping of CRISPR/Cas9-induced indels by distinguishing wild-type and mutant alleles even when no natural restriction site exists at the target locus.
• Plant genetics validation: Applied to the cytokinin receptor gene AHK3 in Arabidopsis thaliana to identify and isolate novel ahk3 null alleles.
• Genotyping workflows: Supports CAPS/dCAPS-based genotyping workflows that combine PCR amplification with restriction enzyme digestion to detect sequence variants.

Methodology:

Implemented in Python; designs derived CAPS (dCAPS) primers by introducing intentional mismatches to create or alter restriction enzyme recognition sites and explicitly accommodates sequences containing small indels.