Multiplexed CRISPR design, which allows for the concurrent and efficient editing of multiple genomic sites, is a powerful tool for complex genetic modifications. However, designing effective multiplexed guide RNA (gRNA) arrays remains challenging due to the exponential increase in potential gRNA array candidates and the significant impact of different target site selections on efficiency and specificity. Recognizing that more stable gRNAs, characterized by lower minimum free energy (MFE), have prolonged activity and thus higher efficacy, we developed MultiCRISPR-EGA, a graphical user interface (GUI)-based tool that employs the Elitist Genetic Algorithm (EGA) to design optimized single-promoter-driven multiplexed gRNA arrays. Computational experiments on gene targets demonstrate that the EGA can rapidly optimize multiplexed gRNA arrays, outperforming other intelligent optimization algorithms in CRISPR interference (CRISPRi) applications, while the GUI provides real-time design progress control and compatibility with various CRISPR-Cas systems. This tool aims to advance the multiplexed gRNA array design process, enabling more efficient and cost-effective genome editing for synthetic biology.
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