RiboJ-assisted non-repeated sgRNA arrays for enhanced CRISPR multiplex genome engineering in Escherichia coli

This study presents the development and application of the RiboJ-aided Multiplexed Base Editing (RAMBE) system and its advanced iteration, the Non-Repetitive RAMBE (NR-RAMBE) system, as efficient and scalable platforms for CRISPR-based genome editing. The RAMBE system leverages RiboJ insulators to process sgRNA arrays, enabling precise and simultaneous editing across multiple genomic loci. Building on this foundation, the NR-RAMBE system introduces engineered ribozymes and non-repetitive sgRNA handles, addressing the inherent limitations of repetitive DNA sequences in CRISPR arrays. This innovation achieves a 23-fold reduction in synthesis complexity, significantly simplifying the construction of sgRNA arrays while maintaining high editing efficiency. These systems were demonstrated to enhance metabolic engineering applications. This integration of advanced RNA processing and non-repetitive design underscores the NR-RAMBE system’s potential to address the growing demands of large-scale genome engineering projects, enabling faster, more efficient, and customizable genetic modifications across a wide range of organisms. • The RAMBE system enables precise, multiplex genome editing in E. coli using RiboJ insulators. • The NR-RAMBE system reduces synthesis complexity 23-fold with diverse ribozymes and sgRNA handles. • We screened 15 ribozymes and sgRNA variants to overcome challenges from repetitive DNA sequences. • Our system increased butyrate production 7-fold and improved acetate utilization in E. coli Nissle 1917. • The NR-RAMBE system serves as a scalable platform for genome editing in synthetic biology and therapeutics. CRISPR-based systems have revolutionized genome editing by enabling precise and efficient genetic modifications. However, achieving multiplex genome editing remains challenging due to limitations in encoding, transcribing, and processing multiple single-guide RNAs (sgRNAs) in repetitive DNA arrays. In this study, we present the RiboJ-Aided Multiplexed Base Editing (RAMBE) system and its advanced iteration, the Non-Repetitive RAMBE (NR-RAMBE) system, designed for efficient and scalable multiplex genome engineering in Escherichia coli . The RAMBE system leverages RiboJ insulators to autonomously process sgRNA arrays, enhancing sgRNA maturation and enabling simultaneous multi-gene editing. We demonstrate editing of up to six endogenous genes in E. coli Nissle 1917 (EcN) in a single step, achieving high target-specific efficiencies of up to 100%, depending on the target and context. This multiplex editing enabled robust butyrate production and improved acetate utilization in engineered EcN strains. Building on this, the NR-RAMBE system incorporates diverse ribozymes and engineered non-repetitive sgRNA handles to minimize sequence repetition. This design reduced synthesis complexity and enabled simultaneous editing of six genomic loci with efficiencies comparable to those of the RAMBE system. The NR-RAMBE system broadens the scope of CRISPR multiplexing by allowing precise and scalable genome editing without labor-intensive sgRNA array assembly, paving the way for diverse large-scale genomic applications.