Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar)

CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies.


Needle preparation and microinjection
Quartz capillaries with filament (QF100-70-10, 10-cm long, 0.7-mm internal diameter and 1.0mm external diameter, Sutter Instruments, Novato, CA USA) were siliconized as follows: 10 mL of Sigmacote (Sigma) was placed into a 50 mL glass beaker and allowed to enter each by capillary action. These were then inverted end to end 6X to ensure that the entire length of each capillary was coated. The capillaries were drained and blot dried on a Kimwipe and then the remaining

Screening and image acquisition
The developing embryos and emerged nymphs were screened for altered eye color using a stereomicroscope. Wild-type GWSS embryos and first-instar nymphs have red-brown eye color 1 , while adults have dark-brown eyes with yellow striations and wings with red pigments in some interveinal spaces (cells) and veins 2 . Eye color is discernable in embryos 5 d after egg deposition.
GWSS nymphs with an edited white gene have white or mosaic eyes, while nymphs with an edited cinnabar gene have red-orange or mosaic eyes. The % embryos and generation 0 (G0) nymphs with mutant eye color were determined. G0 mutant nymphs were transferred to cages with six sunflower plants to complete their developmental cycle 3 (see Methods in manuscript for rearing details).
Photographs of developing embryos and nymphs, as well as the eyes, wings, and bodies of adult wild-type, G0-G4 w and G0-G3 cn GWSS were taken with a Toup Tek Industrial Digital Camera 20MP 1, Sony Exmor CMOS sensor, and 4K UHD Multi-output HDMI Camera coupled to a LEICA M165 FC stereoscope using Toup View camera program. Bright-field images were taken using auto settings for exposures and white balance. Adobe Photoshop 2020 was used to apply moderate changes to image brightness and contrast. Changes were applied equally across the entire image and for all images. analysis, respectively. The purified PCR fragments (300-500 ng) were sequenced by Retrogen Inc (California, USA) using the pJet vector primers. The amplicon sequences from G0, G1, G2, and G3 insects were analyzed via alignments to wild-type GWSS sequences using Cas-analyzer tool 4 and the MUSCLE alignment tool 5 .

Cleavage efficiency of sgRNA in vitro:
The cleavage efficiency of white and cinnabar sgRNAs were evaluated using the Guide-it sgRNA Screening System kit (Takara Bio, Shiga, Japan). sgRNA (50 ng) and Guide-it Cas9 (250 ng) in a final volume of 1.5 μl were assembled at 37°C for 5 minutes. At this time, 30-100 ng of the WT PCR amplicon was added to the tube, followed by 1 μl of 15 x Cas9 buffer, 1 μl of 15 x BSA and 6.5 μl of RNase-free water (total volume of 20 μl). The reaction was incubated at 37°C for one h, followed by 80°C for five min. The digested fragments were fractionated in a 1.5% agarose gel.

Analysis and sequencing of off-target loci
We selected for off-target sequences that contained 3-4 mismatches distal to the seed regions of the three sgRNA target sites with bulge sizes from 0-2 using Cas-OFFinder 6 (Supplementary Table S4b). Genomic DNAs from WhA-D G0 and CnA-F G0 adults prepared as described above were used to characterize the frequency of insertions and deletions at off-target loci. The methods for PCR amplification of off-target regions, Illumina library construction, and amplicon sequencing of loci were performed as described for the w and cn loci in G0 insects. Off-target locus-specific primers and their Tms are provided in Supplemental Table S5b. The complete photograph of the gel shown in Figure 4d. Lane N is the no DNA template control.

Supplementary Figure S4
The complete photograph of the gel shown in Figure 6d. Lane N is the no DNA template contro