Programmable Multiplex Genome Editing: Innovations in CRISPR Effectors, crRNA Engineering, and Delivery Strategies

ABSTRACT: The discovery of CRISPR based technologies has transformed genome engineering and synthetic biology. With advancements in the ability to do multiplex genome editing, it is now emerging as an ideal approach for trait stacking to improve crops, functional genomics, and complex metabolic engineering in various biological systems. This review discusses engineering and optimization of the latest CRISPR effectors for scalable and precise multiplex editing, ranging from well-known systems like Cas9 and Cas12 variants, to newer, smaller variants such as CasMINI, Cas12j2, and Cas12k. We highlight how the emergence of base editors and prime editors enabled efficient editing across multiple loci without double strand breaks. We also elaborate on the expression and processing strategies of crRNA arrays, which are central to any multiplexing approach. These include tRNA-based and ribozyme-mediated methods, synthetic modular designs, and AI-optimized guide RNAs tailored to diverse systems. Additionally, we assess next-generation delivery platforms such as lipid nanoparticles, virus-like particles, and metal-organic frameworks that overcome conventional barriers in in vivo applications. This review provides a critical take on technological advances enabling precise, high-throughput, and programmable multiplex genome editing across biological systems, setting the foundation for future innovations in synthetic biology, crop improvement, and therapeutic intervention in multigene diseases.