## 5 Materials and Methods
The solution was mixed and incubated at room temperature for 5-10 min for Cas9-sgRNA binding, and the solution was kept on ice during microinjection. For knock-out experiments, we used either three sgRNAs or sgRNA#1 alone for microinjections (Table S8, sgRNA target sequences). For knock-in experiments, we only used sgRNA#1 in the microinjection solution. _A. domesticus_ egg microinjection protocol for CRISPR. An egg lay dish with hydrated water crystals was placed into an adult wild type _A. domesticus_ colony for 4 h to receive fresh and non-developed eggs. Eggs were collected and washed using the protocol described above in the \"_A. domesticus_ Colony Maintenance\" section. After washing the eggs from the water crystals, a solution of 70% ethanol (ethanol/deionized water) was used to briefly rinse the eggs for 10-15 s and quickly rinsed with deionized water at least 3 times. The eggs were placed on microinjection slides (Figure S12). To prepare the microinjection slide, a piece of black filter paper was cut into 50 \(\times\) 10 mm pieces and laid on a standard clean glass microscope slide (75 \(\times\) 26 mm). Another two pieces of black filter paper strips, 50 \(\times\) 2 mm, were cut and laid one on the top of the other placed in the middle of the larger piece of black filter paper. Deionized water was added to the filter paper, and eggs were placed on the edge of two sides of the filter paper strips for microinjection. The wet filter prevented the eggs from drying out during microinjection and allowed a backstop to keep the eggs from moving during the procedure. The black paper made visualizing the eggs easier compared to using white filter paper. The needle for microinjection was pulled from Standard Glass Capillaries (1B100-4, World Precision Instruments) using a P-2000 needle puller (Sutter instruments). The following puller apparatus settings were used: Heat: 335 Fil: 4 Vel: 40 Del: 240 Pul: 120. For microinjection, 1 \(\,\)uL of microinjection solution was loaded into the back of the needle with Femtotips (Eppendorf, Hamburg, Germany), and all air bubbles were removed from the liquid in the needle, particularly the tip. The filled needle was then applied to a Femtolet 5247 microinjector (Eppendorf) and air pressure of 4-7 psi was used for holding the solution in place and 9-10 psi for injection. Before injection, forceps were used to gently break the needle tips to achieve a small opening. The injection site for eggs was 2/3 from the smaller end. After injection, the black filter paper containing the injected eggs was transferred from the glass slides to a piece of wet paper towel and kept in a 150 \(\times\) 10 mm petri dish with lid. The dish was placed into a hatch chamber (Modular Incubator Chamber, MIC-101, Billups-Rothenberg, San Diego, CA, USA) with two wet paper towels for 10-12 d.  
Screening for CRISPR knock-out/in and established colonies. To screen for either white eye (Ad vermilion knock-out) or EGFP (EGFP knock-in) expression phenotypes, freshly hatched G\({}_{0}\)_A. domesticus_ were transferred from their egg incubation chamber (described above) into a standard petri dish with lid. The petri dish was then placed on ice to immobilize for 3 min. The crickets where then observed under a fluorescent dissecting microscope with digital camera (Leica M125, Leica Microsystems Inc., Deerfield, IL, USA) for either different eye coloration from wild type in knock-out treatments (using a standard white LED ring light) or using a blue fluorescent light and GFP filter to check for knock-in EGFP expression phenotype. Photos were taken using a QImaging Retiga R6 digital camera. After screening, G\({}_{0}\) crickets with positive knock-out white/vermilion eye (full or partial) phenotype were separated and reared to adulthood in containers described in the house cricket colony maintenance section above. Crickets with wild type eye phenotype from knock-out experiments were not kept.