Utilization of CRISPR/Cas9 to Study Meristem Growth and Shoot Architecture in the Solanaceae

Tomato has become a valuable model for biological, genetic, and functional genomics studies.  Its adoption as a model resulted from readily available resources such as mutant collections, a high quality genome sequence, mapping populations, and efficient transformation methodology.  These resources provided the ideal platform for testing the feasibility of the genome editing technology, CRISPR/Cas9.  We started with a proof-of-concept experiment to target a gene (ARGONAUTE7; SlAGO7) that when function was lost would result in an easily recognizable phenotype (needle-like leaves; wiry) evident as early as the plant regeneration stage following Agrobacterium-mediated transformation of cotyledon explants. The CRISPR construct contained two single guide RNAs (sgRNAs) to produce large deletions to ensure disruption of gene expression.  Twenty-nine independent transgenic lines were recovered and 48% exhibited the expected wiry phenotype, which indicated the successful disruption of SlAGO7.  DNA sequencing of PCR amplicons revealed the CRISPR lines were comprised of a range of mutations including homozygous, biallelic or chimeric small insertions and deletions (indels).  The indels were present at various locations near both sgRNA targets.  Following this success, we have designed more than 40 CRISPR constructs to target genes that were shown through RNAseq analysis to possibly play a role in flowering, stem maturation, and inflorescence branching.  The CRISPR/Cas9 technology has been a powerful tool for our reverse genetics approach to elucidate the roles of these genes.  Genes found to be of interest in tomato will also be targeted in other Solanaceae members to determine cross species effects on meristem development and shoot architecture.