2017 ASHS Annual Conference

The CRISPR/Cas-mediated genome engineering system has received widespread attention as a precise genome editing technique for site-specific gene targeting and gene editing. A major advantage of this system is in its ability to edit multiple target genes simultaneously allowing the researcher to utilize the loss of function approach in understanding the role of these genes. The objective of our research was to develop an efficient plant transformation vector that could result in efficient genome editing in citrus. Three different constructs were evaluated in this study: The first containing a constitutively expressed nuclear targeted Cas9, the second with a constitutively expressed EGFP- Cas9 fusion and third containing a chemical-inducible system with the nuclear targeted Cas9 driven by the LexA operator fused to a 35S minimal promoter. As a proof of concept, the CsTAC1 tree architecture gene was targeted: either with a single gRNA or by the nickase system with two gRNAs targeting sites 10bp from each other. Our results indicated that the chemical-inducible system resulted in the efficient editing of the target gene when coupled with the double sgRNA method. The EGFP- Cas9 fusion construct had the lowest mutation rate among the constructs evaluated. The chemical-inducible system could also minimize the off-target mutagenesis risk associated with constitutive expression of the Cas9.