Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA

Abstract

Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system—a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.

Review: Plant Genome Editing Targeted by RNA-Guided DNA Endonuclease CRISPR/Cas9

“…CRIPSR/Cas9 gene editing methods were applied to other crop plants, tomato [31], maize [32], soybean [33] and sweet orange [34]. Loss of function mutation of ARGONOUTE7 (SALARGO7) resulted in a distinct recognizable phenotype in 48% of T0 plants, first formed leaves with petiole-less leaflets and later formed radialized leaves, and compound flat leaves becoming needle-like or wiry in mostly infertile mature plants [31].…”

Genome engineering of woody plants: past, present and future

“…Jia and Wang [71,72] reported CRISPR/Cas9-based genome editing in the sweet orange, Citrus sinensis. In this latter report, transient expression of plant-codon optimized Cas9 and CsPDS-targeted gRNA disrupted the endogenous CsPDS locus.…”

“…Xcc improves infection efficiencies of Agrobacterium to citrus [72]. In addition, the cauliflower mosaic virus (CaMV) 35S promoter, which is transcribed by RNA polymerase II, was used for the expression of gRNA in sweet orange [71]. RNA polymerase III-transcribed promoters, such as U3 and U6, have more commonly been used to express gRNA.…”

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

“…The gRNAs are expressed using the RNA polymerase III U6 or U3 promoters which restricts the first base of the gRNA to either a G, for U6, or A for U3, for efficient transcription. However RNA polymerase II promoters, which are free of these restrictions, have also been used 7,8 . Different gRNAs induce DNA mutations with different efficiencies, and so it can be important to first validate CRISPR vectors before investing in whole-plant transformations or setting up extensive phenotypic screens.…”