ORIGINAL RESEARCH article
Front. Genome Ed.
Sec. Genome Editing Tools and Mechanisms
Volume 7 - 2025 | doi: 10.3389/fgeed.2025.1571023
This article is part of the Research TopicComputational Methods and AI in Genome EditingView all articles
CATS: A Bioinformatic Tool for Automated Cas9 Nucleases activity comparison in clinically relevant contexts
Provisionally accepted
- 1IRCCS Azienda Ospedaliero Universitaria di Bologna, Bologna, Italy
- 2Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy
- 3Alia Therapeutics, Trento, Italy
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With an overwhelming number of Cas9 nucleases within the genetic engineers' portfolio, picking the right one can be daunting. Because the Cas9 nucleases to compare have usually different requirements in terms of protospacer adjacent motif (PAM) sequences, to do a proper comparison a common target site should be identified. This removes the bias underlying the natural genetic landscape characterizing the target of choice. In this study, we introduce Comparing Cas9 Activities by Target Superimposition (CATS), a novel bioinformatic tool designed to automate the detection of overlapping PAM sequences and identify allele-specific targets resulting from pathogenic mutations. CATS significantly reduces the time and effort required for CRISPR/Cas9 experiment design, making it accessible to a broader range of researchers. By automating the detection step, CATS streamlines the comparison of Cas9 nuclease with different PAM requirements, thus allowing the selection of the most appropriate Cas9 for the target at hand. This can also be performed in the context of genetic variants, extending the tool's use to more clinically relevant settings. One of the key PAM-search parameters of CATS is their existence in close proximity to each other, thus minimizing bias presented by the target sequence composition itself. The most significant strengths of CATS are its automation and speed, its coupling to a continuously updated source of information, and the clean and user-friendly interface, which ensures that all researchers, regardless of their computational expertise, can fully utilize its potential. Furthermore, incorporating ClinVar information, it facilitates targeting disease-causing mutations. Because of all this, CATS supports the implementation of Cas9 applications and the development of therapeutic approaches aiming to treat a given genetic disorder by seamlessly targeting the mutated allele while sparing the healthy-one, ultimately helping the design of more effective therapeutic solutions for genetic disorders.
Keywords: CRISPR/Cas9, PAM, bioinformatics, Genome editing, Bioinformatic software, Pam comparison, Allele-specific
Received: 04 Feb 2025; Accepted: 08 Jul 2025.
Copyright: © 2025 Rocchi, Magnani, Castellani, Carusillo and Tarozzi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Gastone Castellani, IRCCS Azienda Ospedaliero Universitaria di Bologna, Bologna, Italy
Martina Tarozzi, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy
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