Design of DAP Arrays Using tRNA Processing Mechanisms for Multiplex Precision Genome Editing
Background
Base editing and prime editing allow specific bases to be substituted or short sequences to be inserted or deleted without directly cleaving the DNA double strand. As the application scope has expanded from single-gene mutation correction to polygenic disease modeling and functional genomics, there has been a growing need for guide RNA (gRNA) expression systems that can target multiple genomic loci simultaneously.
Conventional multiplex editing methods often require separate promoters for each gRNA or the introduction of additional enzymes and recognition sequences to cleave individual gRNAs from long transcripts. As the number of targets increases, the DNA construct becomes larger and the assembly process more complex. Limitations such as vector payload capacity, gRNA expression variability, and cellular toxicity also remain challenges. In particular, Cas9, unlike Cas12a, cannot process array-transcribed gRNAs on its own, requiring additional separation mechanisms.
Key Discovery
The drive-and-process (DAP) array proposed by the research team is a modular structure that alternately arranges gRNA and transfer RNA (tRNA) units. Once a single transcript is produced, the cell's endogenous tRNA processing machinery cleaves each tRNA, releasing individual gRNAs that bind to Cas9-based editors to recognize multiple targets simultaneously. The key design feature is that no external RNA-cleaving enzymes are required.
This paper is not a study reporting new therapeutic efficacy of DAP technology, but rather a methodology paper that outlines design principles and experimental procedures for implementing multiplex base and prime editing in human cells. Using HEK293T cells as an example, it provides steps for array construction, production, cell delivery, and evaluation of editing outcomes. In foundational research, a 75-nucleotide human cysteine tRNA was applied to simultaneously target up to 31 base-editing sites and up to 3 prime-editing sites.
The DAP array can be reassembled by replacing target gRNAs, and it can be extended to process other RNAs of similar size to gRNAs, such as short hairpin RNA (shRNA). This opens the possibility of designing complex perturbation experiments that coordinate DNA sequence correction and gene expression suppression within a single transcript.
Significance and Prospects
This protocol provides a starting point for researchers to reproduce and optimize multiplex precision editing in the laboratory. It is particularly suitable for creating cell models of complex diseases such as cardiovascular, metabolic, and muscular disorders, where multiple mutations interact, as well as for investigating gene-gene interactions. The short, repeatable modular structure also offers advantages when designing delivery vectors with limited payload capacity, such as adeno-associated virus (AAV) or lentivirus.
However, the fact that the array functions in cultured cells does not immediately imply therapeutic potential. As the array lengthens, synthesis and cloning become more difficult, and the processing level and editing efficiency of each gRNA may vary depending on the target sequence and cell type. When manipulating multiple sites simultaneously, it is necessary to examine not only individual off-target effects but also unexpected editing combinations and cellular functional changes. Validating delivery efficiency, long-term safety, and array stability in primary cells and animal models remains critical for clinical translation.
Recent advancements in base- and prime editing technologies have provided researchers with a wide range of options for introducing precise insertions, deletions, or substitutions into targeted genomic loci. These precision editors have rapidly expanded in application 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.
Pharmaceutical and biotechnology companies can use DAP arrays to introduce multiple disease-related variants into the same cell at once, enabling the creation of complex genetic disease models. For example, they can replicate combinations of risk alleles found in patient populations within cell lines and then compare drug responses and toxicities. In functional genomics research, multiple candidate genes can be edited simultaneously, or some can be suppressed using shRNA to explore synthetic lethal relationships and therapeutic target combinations. To advance toward actual therapeutic applications, it will be necessary to quantify editing efficiency and transcript changes per target in patient-derived cells and to optimize the array length and editor capacity within a single delivery vector.