CaSilico

CaSilico designs CRISPR RNA (crRNA) sequences for Cas12, Cas13, and Cas14 systems to enable specific targeting in genome editing, gene regulation, and nucleic acid diagnostics.

Key Features:

• Comprehensive Rule Integration: Incorporates design rules including mismatch tolerance, self-complementarity, GC content, frequency of cleaving bases near the target site, target accessibility, and protospacer flanking site (PFS) or protospacer adjacent motif (PAM) requirements.
• Flexible Parameter Tuning: Allows adjustment of parameters that influence crRNA selection and off-target identification.
• Reference Compatibility: Supports any reference genome, gene, or transcriptome provided as a FASTA file.
• Multi-Target Design Capability: Detects conserved regions among input sequences to design crRNAs that target multiple sequences simultaneously.
• Support for New CRISPR-Cas Subtypes: Considers newly identified CRISPR-Cas subtypes in its design rules.
• Feature Reporting: Provides a detailed list of features for each candidate crRNA to aid selection.

Scientific Applications:

• Genome Editing: Designs crRNAs for precise targeting in genome editing experiments using Cas12, Cas13, and Cas14.
• Gene Regulation: Enables design of crRNAs for gene regulation studies.
• Pathogen Diagnosis: Supports nucleic acid–based pathogen diagnosis, exemplified by crRNA designs targeting the SARS-CoV-2 genome.
• Multiplex and Conserved-Region Targeting: Facilitates simultaneous targeting of multiple sequences by designing crRNAs against conserved regions among sequence sets.

Methodology:

Computationally integrates specified design rules (mismatch tolerance, self-complementarity, GC content, frequency of cleaving bases near the target site, target accessibility, and PFS/PAM), detects conserved regions among input sequences, performs off-target identification, and operates on reference genomes/genes/transcriptomes provided in FASTA format.