MultiEdit PTG Designer 1.0

Software utilizes polycistronic tRNA-gRNA (PTG)-based multiplex editing system, where the PTG assembly is efficiently and precisely processed into individual guide RNAs (gRNAs) that direct Cas9 to edit multiple chromosomal targets.

For PTG assembly construction, gRNA spacer-specific primers (reverse and forward primers anchor the last and first 12 nucleotides of the 20 bp spacer, respectively) were designed with 4-bp overlap for Golden Gate (GG) assembly. Each module was PCR-amplified (from a plasmid template that contains 77 bp tRNA sequence followed by 86 bp optimized gRNA scaffold sequence) and BsaI-digested in order to create 4-bp overhangs that ligate all the modules together in correct orientation for producing the complete gRNA (spacer + scaffold) without any extra nucleotides.

Terminal parts of PTG assembly contains FokI site that aids in cloning the PTG construct into a Cas9 editor plasmid via FokI digestion that creates compatible 4-bp overhangs. The seamless assembly of multiple PTG modules was wet lab validated and is customizable for deployment in both monocot and dicot plants. As outputs, the software yields primer sequences for each modules, sequences of individual tRNA-gRNA (TG) hybrid modules and the final PTG assembly.