We bought H. zea eggs from Benzon Research (Carlisle, PA) in July 2021. We have maintained this lab strain without exposure to Bt toxins and call it LAB-S due to its sensitivity to Cry2Ab and other Bt toxins27,28,31,41. Larvae were reared on the Southland diet (Southland Products, Inc., Lake Village, AR). All rearing and feeding bioassays were performed at 28°C, 20-40% humidity and 14 h light and 10 h dark. The moths were reared in separate incubators from the larvae and had access to a 10% sugar water solution for food and cheesecloth for egg laying.41.
We used Cry2A.127, a variant of the Cry2Ab protoxin which was prepared, purified and solubilized as previously described.42 and provided by Corteva Agriscience. Cry2A.127 is 98.6% identical to Cry2Ab1 and Cry2Ab2 (9 substitutions out of 633 amino acids) and is referred to herein as Cry2Ab.
Design and synthesis of single guide RNA (sgRNA)
We designed sgRNAs targeting two genes: (1) HzABCA2 encoding the ATP-binding cassette protein ABCA2 (from H. zea genome36); and (2) vermilion (also known as tryptophan 2,3-oxygenase Where HzTO) MG976796.1. The latter gene has previously been shown to cause yellow mutant eye color in CRISPR/Cas9 mutant knockouts of H. zea37. An sgRNA located in HzTO exon 6 and seven sgRNAs located in the 5′ exons of HzABCA2 have been done (HzABCA2 sgRNA 1 and 2 in exon 1; sgRNA 3 and 4 in exon 2; sgRNA 5 in exon 3; and sgRNAs 6 and 7 in exon 4) (Supplementary Table S1). Each sgRNA was adjacent to a corresponding “NGG” PAM site and had no off-target sites based on CRISPOR alignment with H. armigera ABCA2 and To. Each selected sgRNA target sequence was then checked for possible off-target sites by BLAST search in the non-redundant GenBank database. sgRNA DNA templates were synthesized as gBlock DNA (Integrated DNA Technologies, Coralville, Iowa) which contained the T7 RNA polymerase binding site, a 20 bp gene-specific target sequence, and the common sequence of 80 bp stem-loop tracrRNA. DNA templates were used to synthesize gRNA using the HiScribe T7 High Throughput RNA Synthesis Kit (New England Biolabs, Ipswich, MA). Transcribed sgRNAs were treated with DNase I for 20 min at 37°C and purified using RNAClean XP (Thermo Fisher Scientific) according to the manufacturer’s protocol.
In vitro Cas9 cleavage with guide RNAs
To test whether each sgRNA complexed with Cas9 was able to cut amplified by PCR HzABCA2 gDNA in vitro, we used the Guide-it sgRNA screening kit (Takara Bio, Mountain View, CA) as previously published23. We first extracted gDNA from LAB-S 3rd instar larvae using the Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany), which served as DNA template for PCR. Primer pairs 1HzABCA2-5 + 2HzABCA2-3, 3HzABCA2-5 + 4HzABCA2-3 and 5HzABCA2-5 + 6HzABCA2-3 (Supplementary Table S2) were used to amplify PCR products corresponding to exon 1, l exon 2 and exons 3–4, respectively. Each sgRNA was diluted to 50 ng μL−1 in RNase-free water and 1 μL was mixed with 250 ng of Guide-it Cas9 nuclease and incubated at 37°C for 5 min. Fifty ng of each gDNA PCR template was combined with Cas9 reaction buffer, bovine serum albumin, RNase-free water and the appropriate Cas9/sgRNA mix. These were incubated at 37°C for 1 h. Reactions were terminated (80°C for 5 min) and aliquots of each digestion reaction and corresponding negative controls were analyzed by electrophoresis on 1.5% agarose gel stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific).
Micro-injection of embryos
sgRNA targeting HzABCA2 and HzTO were individually complexed with Alt-R Streptococcus pyogenes HiFi Cas9 nuclease V3 (Integrated DNA Technologies), at 50 ng µL−1 of sgRNA at 100 ng µL−1 of Cas9, then incubated at room temperature for 15 min to generate the Cas9-ribonucleoprotein (RNP) complex. The two RNP complexes were combined to create a mixture of HzABCA2 and HzTO RNP, then the mixture was placed on ice and immediately used for injections.
Embryos were collected from LAB-S by placing microscope glass coverslips (24 X 40 mm, Corning Inc., Corning, NY) on wire-mesh lids for multiple cages containing 5 adult males and 5 females for 45 min . Adult H. zea the females naturally attach the eggs to the coverslips and no further manipulation of the embryos was necessary. An IM-300 microinjector (Narishige International USA, Amityville, NY) equipped with an Olympus IMT-2 inverted microscope (Olympus Corporation, Center Valley, PA) was used to inject newly laid eggs (less than one hour old) with about 100–200 picoliters of HzTO + HzABCA2 RNP Solution. Quartz needles (Sutter Instrument Co, Novato, CA) were beveled using a model EG-44 micropipette grinder (Narishige) at an angle of 30° and a rotor speed of approximately 1800 rpm /min or 90% of maximum speed. The needles were filled with 3 µL of the RNP mix using Eppendorf Microloader pipette tips. After injection, the embryo-containing coverslips (n = 177 injected embryos in total) were placed in 100 x 15 mm Petri dishes containing 1% agarose and maintained at 28 °C until G0 newborns appeared.
newly emerged G0 hatchlings were transferred to individual 30 mL translucent polystyrene cups containing 10 mL of Southland diet and reared at 28°C (14:10 L:D) until pupation. Pupae (n = 125) were sexed and transferred to individual 30 ml cups until adults hatched. Two Gs0 adult females showing all-yellow eyes were allowed to mate with two G0 males showing mosaic eyes to generate Yellow-R2 G1 neonates used in diet selection bioassays (below). The rest of the surviving G0 adults (28 males and 27 females) were placed in cages with 8–10 males and 8–10 females and used to generate Hz-R2 G1 hatchlings used for selection (below).
Creation of strains resistant to Cry2Ab Hz-R2 and Yellow-R2
We tested G1 larvae of Yellow-R2 and Hz-R2 unselected strains for Cry2Ab susceptibility using our standard 7-day diet overlay bioassays41. We tested one neonate per well in bioassay trays (BIO-BA-128, Pitman, NJ) on diet treated with 1 μg Cry2Ab per cm2 diet (n=192 for Hz-R2 and n=224 for Yellow-R2) or control diet (n=16) treated with 40 µl of 0.1% Triton X-100 (no Cry2Ab). The trays were covered with Pull N’ Peel lids (BIO-CU-16, Pitman, NJ). Survivors (≥ 4th instar larvae at 7 days) on a diet treated with Cry2Ab were used to further propagate the Hz-R2 and Yellow-R2 strains. We calculated adjusted survival (%) as survival (%) with a diet containing 1 μg Cry2Ab per cm2 divided by survival (%) with untreated diet. We used a two-tailed Fisher’s exact test (http://www.graphpad.com/quickcalcs/contingency1/) to determine if a significant difference has occurred between LAB-S and the G1 of each CRISPR-edited strain in the proportion of live larvae on a treated diet and on an untreated diet.
Cas9-induced mutations in gDNA target regions
To determine if G1 Yellow-R2 and Hz-R2 larvae that survived on the surface of feed treated with 1 μg Cry2Ab per cm2 harbored mutations corresponding to sgRNA target sites, we amplified, cloned and sequenced the DNA of HzABCA2 and HzTO gDNA corresponding to sgRNA target sites. We extracted gDNA separately from LAB-S, Yellow-R2 and Hz-R2 larvae using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Phusion Green high-fidelity DNA polymerase (Thermo Fisher Scientific) was used with oligonucleotide primers (Supplementary Table S2) to amplify specific regions corresponding to both HzABCA2 and HzTO sgRNA target sites. PCR conditions were 98°C for 1 min for 1 cycle; 98°C for 5s, 48°C for 5s and 72°C for 10s for 35 cycles; 72°C for 1 min; and maintain at 16°C. PCR amplicons were run on 1.5% agarose gels and stained with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific). DNA bands were excised, cloned into pJET1.2 vector (Thermo Fisher Scientific) and transformed into One Shot TOP10 Chemically Competent E.coli (ThermoFisher Scientific). Purified plasmid DNA was sequenced by Sanger by Retrogen (San Diego, CA). Multiple sequence alignments were performed using MUSCLE43. The feature film HzABCA2The LAB-S sequence is deposited in the public GenBank database (accession number OP186036.1).
Sensitivity to Cry2Ab of Hz-R2 and LAB-S
We used biological tests superimposed on the diet41 determine the concentration of Cry2Ab required to kill 50% of the larvae (LC50) for Hz-R2 and LAB-S. Newly emerged hatchlings (n = 48 larvae per concentration) were placed in separate wells of bioassay trays. For LAB-S, these contained 0, 0.1, 0.3, or 1 μg Cry2Ab per cm2 power supply, and for Hz-R2 (G4) they contained 0, 1, 3, 10 and 30 μg Cry2Ab per cm2 diet. Trays were maintained at 28°C (14 h light: 10 h dark). After 7 days, we noted live larvae of third instar or more as survivors. We adjusted mortality for control mortality using Abbott’s correction and calculated LC50 using R (v 3.6.3)44 and the publicly available script https://github.com/JuanSilva89/Probit-analysis/commit/2eaaff05da0f89294788bd0bed564e1bf257acf2. The resistance ratio was calculated by dividing the LC50 for Hz-R2 by the LC50 for LAB-S.
Inheritance of Cry2Ab resistance in the Hz-R2 strain: maternal effects, sexual relationship and dominance
We assessed the mode of transmission of resistance to Cry2Ab for Hz-R2 by testing F1 neonates from Hz-R2 X LAB-S reciprocal crosses in diet-overlapping bioassays on 1 μg Cry2Ab per cm2 diet (n = 130 hatchlings per cross) and control diet (n = 32 hatchlings per cross). Survivors (≥ 4th instar larvae) were determined after 7 days of diet exposure and adjusted % survival was calculated by dividing survival with treated diet by survival with diet without Cry2Ab. We evaluated the dominance parameter h which ranges from 0 for completely recessive resistance to 1 for completely dominant resistance45. We calculated hfor Hz-R2 as:h= (F1 survival − S survival)/(R survival − S survival), where S is LAB-S, R is Hz-R2, and F1 is larvae resulting from reciprocal crosses between Hz-R2 and LAB-S.