Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner)

Cotton bollworm is one of the most destructive pests worldwide. Genome editing with the CRISPR/Cas9 technology enables further research of gene function and interaction among different genes in this organism. The two main advantages of this technique are that it has high specificity and is hereditable.

In order to choose the sgRNA targets, go to the CRISPR online website to search for possible guide sites and the exons close to the five prime untranslated region of the gene. Input the exon sequence into the text box and select the target genome to Helicoverpa armigera. Choose the PAM option for 20 BPNGG and leave the other settings on the default parameters, according to the user manuals of the website.

Compare the predicted guide sequences from the software and choose a guide sequence of 20 base pairs containing one or two guanines near the five prime untranslated region with the highest predicted efficiency and fewest mismatches to improve the editing efficiency and reduce off-target editing. For embryo preparation, select 50 healthy individuals from three-day-old males and two-day-old females, and mix them in a clean net cage. Place a piece of moist cotton containing 10%sugar solution in the cage.

Cover the net cages with gauze and fix the gauze with a rubber band. Spray water onto the gauze to keep it moist. Cut a black cloth into an appropriate size and replace the moist gauze with this black cloth to enable free ova-position for 30 minutes.

Paste double-sided tape onto a microscope slide. Using forceps, paste the patches with eggs in a row on the surface of the double-sided tape. Press the margin of each patch to make sure they stick firmly to the tape.

Collect 50 to 100 eggs per microscope slide. Before microinjection, keep the microscope slide on ice to delay the development of embryos. Prepare the needle by pulling a capillary glass using a micro pipette puller as described in the text manuscript.

Grind the needle tip using a micro grinder to a sharp-edged tip. Prepare injection solutions by adding two microliters of commercialized Cas9 protein and sgRNA to RNace-free water in a PCR tube to obtain 10 microliter volume mixture. Mix well by pipetting and put it on ice.

Set the parameters of the electronic microinjector. Load two microliters of the mixture into a needle using a microloader pipette tip, exhausting as much residual air in the tip of the needle as possible. Connect the injection needle to a micromanipulator and ensure a tight connection between the two parts.

Place a slide in a Petri dish and put it on the stage of the microscope. Carefully insert the needle tip into the top hemisphere of an embryo at a 45 degree angle. Press the pedal to deliver the mixture into the embryo, leading to a slight expansion of the embryo.

Retract the needle immediately from the embryo and move the Petri dish with one hand until the next embryo is in proximity to the needle. Inject at least 300 embryos using the same procedure to ensure a sufficient hatching amount. Cover the lid of the Petri dishes after injection.

When the surface color of embryos has darkened, put artificial diet in the Petri dish around the microscope slide. Prepare 24-well culture plates and fill each well to 1/3 of its volumetric capacity with artificial diet. Pick out hatching larva using a small paint brush and transfer them to the 24-well culture plate, such that one well has one larva.

When larva grow to the third instar stage, transfer them to a new glass ductile ethrae. Transfer the newly enclosed G-zero male adults and wild-type female adults into a fresh net cage at an equal ratio. Supply them with 10%sugar solution dropped in cotton balls.

Rear insects using routine methods until the pupation of G-one. Use forceps to carefully remove a hind leg and put each leg in a Lysing Matrix tube. Homogenize the hind leg using a tissue homogenizer.

Extract the genomic DNA of the homogenized sample using a commercial gDNA extraction kit according to the manufacturer's instructions. Amplify the gene segment using genotyping primers and the PCR reaction conditions from the text manuscript. Confirm the genotype with a gene-sequencing service.

Put G-one individuals of the same genotype in one net cage, self cross the G-one progenys and continue to screen using the same methods. The target site of the gene of interest was located in its second exon. This site was highly conserved and the target band fragment of synthesized sgRNA was confirmed using agarose gel electrophoresis.

The mutant detection of a single sgRNA target was performed by sequencing the PCR products from G-one parental units. The effectiveness of using non-overlapping sgRNA pairs across different exons was demonstrated as the large deletion of the mutants was easily distinguished from the wild-type pans. After obtaining a certain number of mutant individuals, electrophysiological or behavioral experiments can be performed to clarify gene function and interaction among different genes.