Variant-aware Cas-OFFinder

In the CRISPR-Cas systems, the guide RNA molecule has the potential to cause the Cas complex to target unintended genomic locations, which are referred to as potential off-target sites. This can lead to incorrect cleavage events. The effectiveness of the CRISPR-Cas system’s targeting is directly influenced by the specific sequence composition of the guide RNA (gRNA) and the presence of PAM in the target DNA sequence. Considering the presence of numerous DNA sequences in the genome that closely resemble the target location, the risk of unintended DNA modifications is significantly heightened. Before initiating any genome editing experiment, it is crucial to predict and assess potential off-target sites.

When predicting potential off-target sites in silico, guide RNA (gRNA) sequences are aligned to a reference genome to identify sites with sequence similarity. However, reference genomes are typically derived from a limited number of individuals and may not fully represent genetic diversity. We present an enhanced version of Cas-OFFinder, a computational tool that extends beyond reference genome-based predictions by incorporating individual genetic variants while maintaining support for unlimited mismatches and flexible PAM sequences, similar to the original Cas-OFFinder.

Variant-aware Cas-OFFinder Webtool

The Variant-aware Cas-OFFinder web tool is designed to assist scientists and researchers in identifying potential off-target sites across an individual’s entire genome. The web tool is developed using SvelteKit for the front-end and FastAPI for the back-end, ensuring a responsive user interface and efficient processing of off-target search queries. PAM types and target genomes are managed through user-editable YAML configuration files, enabling modular customization across different species and nuclease systems. The entire application is packaged using Docker Compose for easy deployment and reproducibility.