Optimizing Gene Editing using a Multi-Purpose Cloning System for Plant Genome Engineering

Genome engineering is a new field of biology that encompasses strategies and techniques for targeted genome modification in any organism. We have developed a comprehensive set of reagents that utilize both TALEN and CRISPR/Cas9 platforms for genome engineering in plant systems amenable to transformation. The reagents are organized into an easy-to-use modular cloning system based on the golden gate assembly protocol that can accommodate different regulatory sequences for either monocots or dicots. This versatile platform can be used for various genome engineering techniques including generating single or multiple gene knockouts and can be multiplexed for gene and chromosome deletions. Moreover, the platform can be integrated with a set of geminivirus-based constructs for precise gene editing through homologous recombination. One of the key advancements of this platform is the incorporation of the Csy4 ribonuclease or tRNA processing enzymes that allows for highly efficient multiplexed gRNA expression. For example, we can routinely generate targeted deletions in up to 6 genes using 12 different gRNAs. In addition, we show that the Csy4 and tRNA systems expressed from a single Pol II promoter are almost twice as effective in induction of targeted deletions as gRNAs expressed from individual Pol III promoters. We also show that mutagenesis can be further enhanced by the incorporation of the TREX2 exonuclease module into any reagent. Our data indicate a 2-fold increase in mutation frequency by CRISPR/Cas9 when employed with TREX2. To date we have successfully tested this platform in tomato, tobacco, Medicago truncatula, wheat, soybean and Theobroma cacao.