CRISP-PTG-Assembler Ver. 1.0: a primer design tool for polycistronic tRNA-gRNA (PTG) assembly for Cas9-based multiplex genome editing in plants.

Multiplex genome editing using the CRISPR/Cas9 system allows simultaneous modifications at several genomic sites, offering great potential for crop improvement. Among various approaches, the polycistronic tRNA-gRNA (PTG) system is widely adopted due to its use of the host's native tRNA processing machinery, enabling the generation of multiple sgRNAs from a single transcript without the need for expressing any foreign RNA processing enzymes or ribozymes. However, designing the complete set of primers suitable for performing in vitro PTG assembly is complex and needs expertise, as a single mistake can lead to complete failure of the assembly process or subsequent editing. To overcome this challenge, we developed CRISP-PTG-Assembler Ver. 1.0, a user-friendly tool that takes only (i) 20-nucleotide sgRNA spacers and (ii) 4-nucleotide joiners as inputs; and produces colour-coded outputs in forms of (i) Primer-set required for complete PTG assembly, (ii) Primary PCR Amplicons, (iii) Overlap-Extension PCR Amplicons and (iv) Expected PTG assembly, for easy interpretation and construct making. Our novel assembly approach provides flexibility in sticky-end choice during golden gate ligation and ensures the fidelity of component sgRNAs in the PTG assembly by buffering against ligation errors (~ 1.5-40%) that may occur during the Golden Gate assembly process, thereby safeguarding the functionality of the in vivo-generated individual sgRNA molecules. We validated its effectiveness by editing two loci of the matrix metalloproteinase 1 gene in rice and demonstrated its applicability across various plant systems. With an intuitive interface and robust features, CRISP-PTG-Assembler empowers researchers of all levels to effectively implement PTG-based multiplex genome editing in plants.