Effective CRISPR-Cas9 Method for Citrus Plants Developed

Citrus is one of the most difficult plants to improve through traditional breeding due to its complex reproductive biology. But the technological advancement in genome editing may help overcome this challenge to develop better varieties of this globally important fruit crop.

Protoplasts are often used for the development of genetically modified plants by the in vitro integration of rDNA, either plasmid-based or linear, into the plant genome. And new methods like genome editing provide options to increase protoplast transformation efficiency to produce more viable cells with integrated donor DNA in their genomes. Thus, American scientists developed a protocol for protoplast transfection using Lipofectamine LTX Reagent with PLUS Reagent to guarantee donor DNA delivery in sweet orange (Citrus sinensis L. Osbeck) protoplasts with low cytotoxic effects on the cells. The new protocol was meant to successfully edit the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, which is a negative regulator of systematic acquire resistance (SAR) that controls the proteasome-mediated degradation of Nonexpressor PR1.

Using the new protocol, a CRISPR-Cas9 construct containing a guide RNA that targeted the CsNPR3 gene was transfected into citrus protoplasts to produce nine genome-edited sweet orange plants. The plants exhibited the downregulation of CsNPR3 expression and upregulation of CsNPR1. The protocol proved to be a viable option for the successful delivery of donor DNA and successful genome editing in citrus to improve plant growth and increase biotic and abiotic stress tolerance.

Learn more about the new method in the study published by Plant Methods.