Web-based tool improves CRISPR accuracy by identifying off-target effects across genetic variations

The genome publishing technologies like CRISPR-CAS9 have transformed biology, medicine and agriculture, but concerns remain concerning involuntary modifications on the DNA sites outside the target. These excluding target effects can cause harmful mutations and are difficult to predict, because they depend not only on the guide RNA but also on the unique genetic composition of an organism. Most existing prediction tools are based on a standard reference genome, ignoring key variations such as SNP, insertions and deletions that differ between individuals and alleles.

To solve this problem, the researchers led by Professor Jeongbin Park of the National University of Pusan ​​have developed Cas-Offinder, Aware-Aware, a web tool that improves the prediction of the site outside the target taking into account the individual genetic variation. The co-prirs Abyot Melkamu Mekonnen (candidate Ph.D.) and Kang Seong (undergraduate student at the time, now a master’s degree in Kaist), who conducted the study while during the Pusan ​​National University, said: “It is very rewarding that the technology we have developed can be practical to correct the individual genes.”

The tool was tested using the human genome and the pepper cultivars. Their results were published online on May 8, 2025 in the journal Nucleic acid search.

The Cas-Offinder tool, aware of the variant, offers a significant front step in the personalized edition of the genome by incorporating genetic diversity directly into the target forecasts. He accepts VCF files to a single sample and supports GPU acceleration, allowing application beyond standard model organisms. By reconstructing the sequences of the genome specific to alleles, the tool performs an analysis at the level of the haplotype, offering a detailed and precise image of the potential target effects.

“In my opinion, the genome editing tools should be as individualized as the genomes they target,” said Professor Park. “Our tool sets the basics of more precise and personalized editing strategies.”

The team tested the tool on the genomes of human and sweet pepper (Capsicum annuum) using public and specific cultivar sequencing data. In humans, he revealed out -of -target sites on chromosome 10 absent from the standard reference genome. In sweet pepper, he identified distinct targets specific to alleles, useful for plant farming.

Compared to existing tools as a case Offinder and Crispritz, Cas-Offinder Variant has systematically identified unique targets caused by small variants such as insertions and deletions. Although it does not yet detect large structural variants, it supports 557 species and 40 PAM types, and is fully customizable via YAML files.

“The precision edition of the genome requires precision tools”, notes Professor Park. “We believe that Cas-Offinder, Away Aware, will play a central role in the development of safer and more efficient CRISPR therapies and agricultural applications.”

The tool is available both as a friendly web interface and a command line version. Its source code, reference tools and data example is available for free on GitHub and Zenodo. Although its analysis at the haplotype can slightly slow performance compared to previous tools, this compromise allows greater precision and reliability in the search for genome editing.