Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg

MINORg is an offline gRNA design tool that gen-erates the smallest possible combination of gRNA capable of covering all desired targets in multiple non-reference genomes. As interest in pangenomic research grows, so does the workload required for large screens in multiple individuals. MINORg aims to lessen this workload by capitalising on sequence homology to favour multi-target gRNA while simulta-neously screening multiple genetic backgrounds in order to generate reusable gRNA panels. We demon-strated the practical application of MINORg by knock-ing out 11 homologous genes tandemly arrayed in a multi-gene cluster in two Arabidopsis thaliana lin-eages using three gRNA output by MINORg. We also described a new PCR-free modular cloning system for multiplexing gRNA, and used it to knockout three tandemly arrayed genes in another multi-gene clus-ter with gRNA designed by MINORg. Source code is freely available at https://github.com/rlrq/MINORg.

Automated summary

MINORg is an offline gRNA design tool that generates the smallest combination of gRNA to cover desired targets in multiple non-reference genomes. By utilizing sequence homology and screening multiple genetic backgrounds, MINORg aims to reduce workload and create reusable gRNA panels. Practical application of MINORg was demonstrated by knocking out homologous genes in Arabidopsis thaliana and implementing a PCR-free modular cloning system. The source code is freely available at https://github.com/rlrq/MINORg.