Constructing Drive-and-Process (DAP) CRISPR Guide RNA Arrays for Multiplexed Base- and Prime-Editing.

Advancements in base- and prime editing technologies in recent years have offered researchers a plethora of options for introducing precise insertions, deletions, or substitutions into targeted genomic loci. These precision editors' applications have rapidly expanded to the modeling and treatment of polygenic diseases, as well as into the growing field of functional genomics. This has created a need for compact, modular expression systems capable of producing multiple guide RNAs (gRNAs) from a single transcript while preserving high editing efficiency. To address this, we have developed the drive-and-process (DAP) array, a modular architecture composed of alternating gRNA and tRNA units. The DAP array design exploits the cell's endogenous tRNA processing machinery to cleave each tRNA from the array and release individual gRNAs, thereby enabling simultaneous editing at multiple loci following hybridization with Cas9. Here, we outline key design considerations and experimental steps required for the construction and deployment of DAP arrays as multiplex base- or prime editing tools in human cells (e.g., HEK293T). We also highlight how the DAP array can be leveraged to enable efficient processing of gRNAs along with other RNAs of similar size, such as shRNA, potentially broadening its usage in addressing complex biological questions and therapeutic applications that require coordinated genetic perturbation.