Lygus hesperus rearing
A laboratory colony of L. hesperus, collected in Maricopa, AZ, served as the source of insects. Adults were maintained in 0.03 m3 screened plastic cages containing shredded paper that were housed in an environmental chamber set at 27 ± 1 °C, 40–60% RH, and a 14:10 (L:D) h photoperiod. Fresh green beans (Phaseolus vulgaris L.), an artificial diet pack53, and a bottle of water with a wick were used to nourish and hydrate the colony and were replaced as needed.
Embryonic RNAi
dsRNA targeting LhCd (MH806847), LhCn (MH806848), and LhW (MH806842) was produced as described in Brent and Hull (2019) to a concentration of 1 µg/µl. In brief, ~ 500-bp products were PCR amplified from validated plasmid DNAs harboring LhCd, LhCn, or LhW using T7 promoter containing primers (Supplementary Table S4). PCR products were purified then used as templates for in vitro transcription using a MEGAscript RNAi kit (Thermo Fisher Scientific). dsRNAs corresponding to the fluorescent protein gene venus, which was injected as a negative control, were similarly generated.
Gel packs, made of Parafilm M (Pechiney Plastic Packaging, Chicago, IL) and filled with carrageenan (1.25% w/v), were provided to L. hesperus as an oviposition substrate for one hour. The gel was removed from the packs and the egg-embedded parafilm sheet retained. The parafilm was stretched to release the eggs, which were then transferred to a moistened filter paper using a wet, fine-tip paintbrush. Eggs were aligned in 4 rows of 10 for a total of 40 eggs per treatment per experiment (LhCd, LhCn, LhW, and venus) and then gently covered with a No.1, 24 × 40 mm coverslip coated with permanent linerless double-sided Scotch tape (3 M, Maplewood, MN). The bare side of the coverslip was mounted on a glass slide with double-sided tape that adhered to the corners of the coverslip. The slide was placed on a Leica DMIL scope (Allendale, New Jersey). An IM 300 Microinjector (Narishige International USA, Amityville, NY) with a quartz needle loaded with dsRNA was used to inject the embryos at the posterior pole. Needles were produced by pulling capillary tubes with filament using a P-2000 needle puller (Sutter Instrument, Novato, CA) with the following two-line program: Line 1) Heat = 850, Filament = 5, Velocity = 25, Delay = 128; and line 2) Heat = 700, Filament = 5, Velocity = 50, Delay = 150. Needles were beveled using a Model EG-44 micropipette grinder (Narishige) at a 30° angle and an approximate rotor speed of 1800 rpm or 90% of the maximum speed. Needles were backfilled using a Microloader tip (Eppendorf, Enfield, CT). Following injection, coverslips with eggs were placed in a covered plastic petri dish containing 1% agarose (1 g agarose in 100 ml of distilled water), which was sealed with parafilm and placed in a growth chamber with the same settings as the laboratory colony. Images of eggs were taken 5 days post-injection using a Nikon SMZ18 microscope equipped with a Nikon D5-Ri2 camera (Nikon Instruments Inc., Melville, NY).
To confirm knockdown of targeted transcripts, expression of LhCd, LhCn, and LhW was measured by semi-quantitative RT-PCR, using actin (GDHC01004191) as a loading control. Total RNA was isolated from four replicated groups of three eggs using a Quick-RNA Microprep kit (Zymo Research, Irvine, CA). RNA quality and quantity were assessed using the Take3 module on a Synergy H4 Hybrid Multi-Mode Microplate Reader (Biotek Instruments, Winooski, VT). Total RNA (250 ng) was treated with DNase I (New England Biolabs, Ipswich, MA). cDNAs were generated from 250 ng RNA using a SuperScript III First-Strand Synthesis System (Life Technologies) and custom-made random pentadecamers (Integrated DNA Technologies, San Diego, CA). Fragments (~ 500 bp) of the genes of interest were amplified in a 20 μl reaction volume using SapphireAmp Fast PCR Master Mix (Clontech Laboratories Inc., Mountain View, CA) and primers listed in Supplementary Table S4. PCR conditions consisted of an initial denaturation at 95 °C for 2 min followed by 35 cycles of 95 °C for 20 s, 56 °C for 20 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. Gel images were obtained using an Azure 200 Gel Imaging Workstation (Azure Biosystems, Dublin, CA) and processed in Adobe Photoshop v21.2.12 (Adobe Systems Inc., San Jose, CA). Independent RNAi experiments were repeated three times.
Design and synthesis of sgRNAs
sgRNAs were designed using LhCd and LhCn with a focus on identifying guide sites near the 5′-end of the gene using CRISPOR54. Both sets of gene-specific sgRNAs were designed in proximity to one another; LhCd1 and 2 are separated by 8 nucleotides while LhCn1 and 2 are separated by 5 nucleotides. sgRNAs were screened for potential off-target sites by BLASTn of the 20-bp target sequence and the PAM sequence against the L. hesperus taxid 30085 database. Potential off-target sites were determined by comparing the BLASTn hit sequences that exactly matched the 3′ end of each sgRNA and the PAM sequence.
Double-stranded gBlock DNA fragments were synthesized by Integrated DNA Technologies (Coralville, Iowa), with each containing a T7 RNA polymerase binding site (5′-TAATACGACTCACTATA-3′), the 20-bp L. hesperus-specific target region (Supplementary Table S5), and the 80-bp common stem-loop tracrRNA sequence (5′-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3′). Each gBlock was used as a template for sgRNA synthesis using the HiScribe T7 High Yield RNA synthesis Kit (New England Biolabs). Transcribed sgRNAs were purified using RNAClean XP (Thermo Fisher Scientific) following the manufacturer’s protocol.
Creation of CRISPR/Cas9 eye pigmentation mutant strains
The experimental design included two independent injection groups; the first injections used a Cas9 protein with a nuclear localization signal (PNA Bio, Newbury Park, CA), whereas the second set used the Alt-R Streptococcus pyogenes HiFi Cas9 nuclease V3 (Integrated DNA Technology, Coralville, Iowa). The injection mixture consisted of the RNP complex of Cas9 (300 ng/µl) with two sgRNAs each at 150 ng/µl or a total of 300 ng/µl. Each sgRNA was preincubated with Cas9 at room temperature for 15 min and both solutions of RNP were combined to make the injection mixture. Negative controls include “no injection” and water only. Embryos were prepared and injected as previously described for the RNAi experiments. A total of 80 eggs per treatment (LhCd1 + 2, LhCn1 + 2, non-inject) were injected in the first experiment and the second experiment included 160, 80, 80, and 20 eggs for LhCd1 + 2, LhCn1 + 2, non-inject, and water, respectively.
Six to nine days post-injection, 1st instar nymphs that hatched were collected into a 355 ml mesh lidded paper cup and reared to adulthood under rearing conditions identical to those outlined above. Pairs from each subsequent generation were mated using the crossing schemes shown in Fig. 3. Card cross 1 (× 1) between a mutant male and a mutant female produced 19 G1 males and 15 G1 females. Card × 2, which also crossed mutants of both sexes, generated 9 G1 males and 12 G1 females. All G1 males from Card × 1 were group-mated with females from Card × 2 and vice versa to generate G2. To perpetuate the mutant line, G2 with red eyes were selected and transferred to new cages. In addition, Card × 3–5 each consisted of one mutant male and four wild type females. The G1 progeny with wild-type eye color from Card × 3–5 were combined and group-mated to generate a mix of wild-type and mutant eye color G2 progeny. From the resulting G2 progeny, 19 males with the mutant eye phenotype were crossed with 38 G2 mutant females from the Card × 1 and × 2 lines. Mutant progeny arising from this group-mating, as well as individuals from the ongoing mutant lines of Card × 1 and × 2 formed the Card colony.
To generate the Cinn colony, two mutant G0 females were crossed with two wild-type males to produce G1 progeny with wild-type eyes. Of these, three females and four males from Cinn × 1 were backcrossed with three males and 13 females from Cinn × 2, respectively. G2 progeny with the mutant eye phenotype were selected to perpetuate the Cinn colony. Mutant colonies were reared in 355 ml paper cups covered with mesh lids with up to 50 individuals. To prevent overcrowding in larger groups of > 50 individuals, 1.89 L paper cups were used55. Fresh green beans and sunflower seeds were provided twice a week. Diet and oviposition carrageenan packs were placed into rearing cups one week post-adult emergence.
Sequencing LhCd and LhCn from mutant eye pigment strains
Representative insects from CRISPR strains that displayed altered eye pigmentation were collected at G0, G1, G2, and/or G3 and stored at -80 °C in RNALater (Invitrogen, Carlsbad, CA). gDNA was extracted using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). Total RNA was extracted using TRI Reagent following the manufacturer’s protocol. Total RNA was treated with DNase I (Thermo Fisher Scientific) and cDNA was synthesized using a SuperScript IV First-Strand Synthesis kit (Invitrogen). LhCd and LhCn were PCR amplified from gDNA and cDNA using a Phusion High-Fidelity PCR kit (Thermo Fisher Scientific) with gene-specific primer pairs (Supplementary Table S4) and thermocycler conditions of 1 cycle at 98 °C for 30 s; 35 cycles at 98 °C for 5 s, 60 °C for 10 s, 72 °C for 5 s; and 1 cycle at 72 °C for 5 min. PCR products were cloned into pJET1.32/blunt vector (Thermo Fisher Scientific) and transformed into One Shot OmniMAX 2 T1 Chemically Competent E. coli (Thermo Fisher Scientific). Multiple clones (n = 4–10) from each transformation reaction were Sanger sequenced (Retrogen Inc., San Diego, CA).
