Riboj-Assisted Non-Repeated Sgrna Arrays for Enhanced Crispr Multiplex Genome Engineering

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 a novel approach with the RiboJ-aided Multiplexed Base Editing (RAMBE) system and its advanced iteration, the Non-Repetitive RAMBE (NR-RAMBE) system, designed for highly efficient and scalable multiplex genome engineering. The RAMBE system leverages RiboJ insulators to autonomously process sgRNA arrays, significantly enhancing sgRNA maturation and enabling simultaneous multi-gene editing. We demonstrate its efficacy by achieving robust butyrate production and improved acetate utilization in Escherichia coli Nissle 1917 (EcN) through the efficient editing of up to six endogenous genes in a single step. Building on this foundation, the NR-RAMBE system incorporates diverse ribozymes and engineered non-repetitive sgRNA handles to address challenges associated with repetitive DNA sequences. This innovation achieves up to a 23-fold reduction in synthesis complexity of sgRNA arrays compared to RAMBE approaches, while maintaining comparable editing efficiencies. The NR-RAMBE system enables precise, simultaneous editing of multiple genomic loci without requiring labor-intensive assembly methods for sgRNA arrays, significantly broadening the scope of CRISPR multiplexing. These advancements collectively establish the NR-RAMBE system as a transformative tool for overcoming critical limitations in multiplex genome editing, paving the way for large-scale genomic engineering and its diverse applications.

Keywords: Base Editing, Multiplex Genome Editing, RiboJ-aided Multiplexed Base Editing (RAMBE), Non-Repetitive RAMBE (NR-RAMBE), Metabolic engineering, Synthetic biology