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Q&A ViewAttendees 1

Plant-Insect Ecosystems

10-Minute Paper

P-IE: Vectors of Plant Pathogens

Highly efficient gene editing in Homalodisca vitripennis: Perspectives on agricultural pest management

Wednesday, November 3, 2021
2:30 PM - 2:42 PM MT
Location: Colorado Convention Center, Meeting Room 103-105
In-Person
Homalodisca vitripennis (glassy-winged sharpshooter, GWSS) is an important agricultural pest that infests over 100 different plant species. GWSS is an efficient vector of the bacterium Xylella fastidiosa, the causal agent of Pierce’s disease of grapevine, Citrus Variegated Chlorosis, and other important diseases. Through species-specific alteration of precise genome locations, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) is a powerful genetic technology that can enable innovative, sustainable and environmentally friendly management of insect pests. Our initial goal was to achieve CRISPR editing phenotypically recognized genes (white-w and cinnabar-cn) to generate knock-out and knock-in mutations in GWSS. Cas9 protein and sgRNAs were delivered by microinjection into developing embryos, resulting in over 60% mutagenesis for both loci with mosaicism of eye color clearly observed in G0 embryos, nymphs and adults. Sequencing of the w and cn target regions demonstrated the inheritance of mutant alleles across the G0 to G2 generations. Reciprocal pair mating indicated that both loci were autosomal. A CRISPaint strategy (non-homologous end-joining DNA repair following CRISPR/Cas9 mutagenesis) was used to integrate a reporter gene, mCherry, under the control of the whitefly polyubiquitin promoter into the cn gene. The frequency of cn-mutant eyes was 60%. PCR analysis showed integration of mCherry in 83% of the mutants. However, no mCherry fluorescence was detected, indicating that the whitefly promoter does not function in GWSS. Our study demonstrates a very high-frequency gene editing in GWSS and the potential of this insect to become a model for developing new genetic control strategies.