RiboJ-Assisted Non-Repeated sgRNA arrays for enhanced CRISPR multiplex genome engineering in Escherichia coli

Abstract

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.