CRISPy-web 3.0: A unified platform for multi-modal guide RNA design for CRISPR and TnpB genome editing applications.

CRISPy-web is an interactive web-based platform designed for rational guide RNA design in CRISPR-Cas9-based genome editing systems. Here, we present CRISPy-web 3.0 (https://crispy.secondarymetabolites.org/), an upgraded and versatile version that extends its functionality beyond classical Cas9-based systems. This version integrates support for diverse genome editing systems, including Cas9, CRISPR interference (CRISPRi), and TnpB/ωRNA. The redesigned interface enables users to toggle between multiple editing modes, select target regions such as ORFs or 5' UTRs, and visualize strand orientation, off-targets, and predicted mutation outcomes. It also incorporates scoring systems that evaluate guide RNA efficiency and specificity based on mismatch tolerance, positional context, and PAM requirements for CRISPR-Cas9-based applications. With an improved user interface design, enhanced backend scalability, and modular support for customizable inputs, CRISPy-web 3.0 provides a comprehensive and extensible platform for guide RNA design, enabling genome editing across a broader range of prokaryotic systems, including both CRISPR and TnpB.