Staff at Pusan National University

CRISPR-based gene editing holds great promise, but off-target effects remain a major concern, especially across diverse genetic backgrounds. A new study presents a web-based tool that enhances off-target site prediction by incorporating individual genetic variants. Developed using the human genome and pepper plant cultivars, the tool improves accuracy at the haplotype level. The user-friendly login-free platform offers researchers a powerful way to personalize and safeguard genome editing applications across fields.

Genome editing technologies like CRISPR-Cas9 have transformed biology, medicine and agriculture, but concerns remain about unintended edits at off-target DNA sites. Those off-target effects can cause harmful mutations and are difficult to predict because they depend not only on the guide RNA but also on an organism’s unique genetic makeup. Most existing prediction tools rely on a standard reference genome, ignoring key variations like SNPs, insertions and deletions that differ across individuals and alleles.

To address that issue, researchers led by Professor Jeongbin Park from Pusan National University developed Variant-aware Cas-OFFinder, a web-based tool that improves off-target site prediction by accounting for individual genetic variation.

Co-first authors Abyot Melkamu Mekonnen and Kang Seong, who conducted the study while at Pusan National University, said, “It is very rewarding to know that the technology we developed can be of practical help in correcting individual genes.”

The tool was tested using the human genome and pepper cultivars. Their findings were published online May 8, 2025, in the journal Nucleic Acids Research.

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The Variant-aware Cas-OFFinder tool offers a significant step forward in personalized genome editing by incorporating genetic diversity directly into off-target predictions. It accepts phased single-sample VCF files and supports GPU acceleration, allowing application beyond standard model organisms. By reconstructing allele-specific genome sequences, the tool performs haplotype-level analysis, offering a detailed and accurate picture of potential off-target effects.

Park said, “In my view, genome editing tools should be as individualized as the genomes they target. Our tool lays the groundwork for more accurate and personalized editing strategies.”

The team tested the tool on human and sweet pepper genomes using public and cultivar-specific sequencing data. In humans, it revealed off-target sites on chromosome 10 absent from the standard reference genome. In sweet pepper, it identified distinct allele-specific off-targets, useful for plant breeding.

Compared with existing tools like Cas-OFFinder and CRISPRitz, Variant-aware Cas-OFFinder consistently identified unique off-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 through YAML files.

“Precision genome editing requires precision tools,” said Park. “We believe Variant-aware Cas-OFFinder will play a central role in developing safer, more effective CRISPR therapies and agricultural applications.”

The tool is available both as a user-friendly web interface and a command-line version. Its source code, benchmark tools and example datasets are freely available on GitHub and Zenodo. While its haplotype-level analysis can slow performance slightly compared to earlier tools, that trade-off enables greater accuracy and reliability in genome editing research. Visit www.pusan.ac.kr/eng/Main.do for more information.

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