Informed sgRNA Design for Genome Editing in Medicinal Cannabis

Matchett-Oates, L.1,2, Braich, S.1,2, Rochfort, S.J.1,2, Spangenberg, G.C.1,2 and Cogan, N.O.I.1,2

1Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Bundoora, Victoria 3083, Australia

2School of Applied Systems Biology, La Trobe University, Bundoora, Victoria 3086, Australia

Cannabis has been used for industrial, recreational and medicinal use for in excess of 4,000 years. However, to date no successful attempts at editing genes involved in cannabinoid biosynthesis have been reported. The development of a best practices approach for the design and implementation of genome editing technologies in cannabis to target all genes involved in cannabinoid biosynthesis is needed. A large population of reference genomes were sequenced and mined to determine copy number variation and associated SNP variants for optimum target edit sites for genotype independent editing. Copy number variation and highly polymorphic gene sequences exist across specific genes in the genome making genome editing using CRISPR and Zinc Fingers more difficult. Evaluation of alleles and gene copy number variants was determined through nucleotide and amino acid sequence alignments with comparative analysis performed. From the determined gene copy number and the presence and location of SNPs, multiple CRISPR design tools were used to design sgRNA targeting every gene, accompanying allele and homologs throughout all involved pathways. The generated catalogue of constructs will enable a wide array of knockouts to be created for functional genomics studies. Universal sgRNA were designed for highly homologous sequences using MultiTargeter and visualised using Sequencher, creating unique sgRNA avoiding SNP and shared nucleotide locations targeting optimal edit sites. Using this framework, the approach established has wider applications to plant species with a range of ploidy level or highly homologous gene sequences.