Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems

Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture. • Multiplex editing enables more complex control over biological systems. • Precise editing at multiple loci advances therapeutic and agricultural applications. • Guide RNA arrays reduce delivery cargo size and promote uniform multiplex edits. • Emerging non-CRISPR systems extend multiplexing to large-scale genome rewriting.