Development of Multiplexed Orthogonal Base Editor (MOBE) Systems
In recent years, with the rapid development of gene editing tools, especially the introduction of the CRISPR-Cas9 system, it has become possible to precisely modify specific DNA sequences. Currently, the introduction of single-nucleotide variants (SNVs) as a powerful tool for studying gene function and disease associations is particularly important in the field of precision medicine. Existing gene editing tools have shown significant efficiency in targeted editing, but they often encounter problems when introducing multiple point mutations simultaneously, necessitating the use of multiplexed editing systems to enhance efficiency.
The research team led by Quinn T. Cowan, Sifeng Gu, Wanjun Gu, Brodie L. Ranzau, Tatum S. Simonson, and Alexis C. Komor from the University of California San Diego published an innovative study in the journal Nature Biotechnology. They successfully developed a multiplexed orthogonal base editor (MOBE) system for precise co-editing on the same DNA strand.
The MOBE system employs RNA aptamer-protein systems for orthogonal multiplexing of guide RNA, addressing the issue of RNA-guided interactions in existing gene editing systems, thus improving editing specificity and efficiency. In the study, two main base editors—deaminases (such as cytosine BE and adenine BE)—were selected and optimized, resulting in the establishment of four different MOBE systems. These systems achieved a precise co-editing rate of up to 7.1% in human cells without enrichment or selection strategies. Using fluorescence enrichment strategies, the co-editing rate could be increased to 24.8%.
In this study, the interaction between RNA aptamers and proteins (coat protein) was used to directly recruit DNA modifying enzymes to the required guide RNA, achieving accurate multi-point base conversion under strict orthogonality control. By expanding the use of PAM (Protospacer Adjacent Motif) and high-fidelity Cas9 variants, the MOBE system demonstrated significant potential in improving applicability to different cell types, expanding targeting range, and enhancing editing quality. This research significantly advances the application of multi-point SNV models in human disease studies, providing new insights and tools for the study of the molecular mechanisms of polygenic diseases and the functional analysis of unknown variants.
Additionally, following the successful development of this editing system, the research team evaluated its orthogonality and found that the MOBE system showed an average 19-fold improvement in orthogonality compared to traditional non-orthogonal systems. The new tool also represents a modular design that can be easily used with other CRISPR-Cas9 variants (such as ncas9-ng, ncas9-spry, or hifi-ncas9), demonstrating lower off-target DNA and RNA editing activities compared to their parent components.
The applicability and efficiency of the MOBE system were validated in multiple cell types, including HeLa cells and SH-SY5Y cells. The MOBE system not only achieved significant breakthroughs in gene editing technology but also holds broad application prospects for multi-target, efficient, and low-cost editing in genomic projects. This is particularly promising for research and clinical applications in complex genetic diseases and gene therapy.
The MOBE system developed in this study provides an effective tool for achieving multi-point base conversions, enhancing researchers’ ability to study specific variant functions in complex genetic backgrounds. This technological breakthrough has extensive potential applications in precision medicine, artificial genome design, and gene therapy, laying a solid foundation for further improvement and optimization of related technologies in the future.