Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

For the first time we have established a modification of the genes coded for ionotropic GABAA and metabotropic glutamate receptors in subsets of neurons in the adult honey bee brain. CRISPR-Cas9 gene editing can be used to modify one or multiple genes in adult bee brains to explore their roles in learning and memory under controlled laboratory conditions. This method can be used to study the function of specific proteins in adult bees, as well as to test the specificity of antibodies against their corresponding proteins.

Demonstrating the procedure with Irina Sinakevitch will be Lev Kurtzman and Hyun Choi, former undergraduates from my laboratory. To test the expression of RDL and mGlutR1 by immunocytochemical analysis after CRISPR-Cas9 injection, first, use an online CRISPR-Cas9 tool to design the guides using the guide DNA sequence of apis mellifera, RDL, and mGlutR1. To prepare guide RNA complex formations for each guide RNA, label one test tube for each guide with name of the guide RNA, and add 92 microliters of nucleotide-free buffer, four microliters of 100 micromolar fluorophore, labeled transactivating CRISPR RNA, and four microliters of the appropriate guide RNA solution to each tube.

When all of the materials have been added, mix the solutions gently and spin the tube contents in a bench-top centrifuge for five seconds to sediment the solution. Then, keep the solutions to 95 degrees Celsius for five minutes to create a guide RNA complex followed by 10 minute cool-down at room temperature. To prepare the ribonucleoprotein complex formations add six microliters of each guide RNA solution and six microliters of 0.5 micrograms per microliter SP-Cas9 nuclease V3 to the appropriately labeled tubes.

After gentle mixing, incubate the solutions at 37 degrees Celsius for 10 minutes To make RNP ribonucleoprotein mixtures for injection, at the end of the incubation add four microliters of each ribonucleoprotein to the appropriate RDL. To make a control, no-guide RNA solution mix four microliters of tracer RNA with 92 microliters of buffer and four microliters of water. After the addition of water, keep the solution for five minutes at 95 degrees Celsius and allow the mixture to cool down until it reaches room temperature.

Then, mix six microliters of the no-guide RNA solution with six microliters of 0.5 micrograms per microliter Cas9 nuclease V3.For ribonucleoprotein mixture injection, after capturing individual bees in vials with a small hole in the caps, immobilize the bees for no more than three minutes on ice and secure each bee into previously-prepared metal holders with duct tape. With the back thorax, wings and head exposed. Use a five milliliter syringe to feed the bees with a one molar sucrose solution until they are no longer hungry, and place the secured bees in a box with a wet paper towel for humidity.

Next, place wax on the inside of the lids of two 35 milliliter petri dishes and place the bottoms of the dishes onto the wax. Inject one molar sucrose between the cover and bottom of each dish, and place one feeding petri dish into white box with one glass slide and place the other into black box with one glass slide. Then, place a small comb into each box and load a microinjection system with the RDL CRISPR-Cas9 mixture.

For RDL CRISPR-Cas9 injection, inject the median ocelli of eight bees with 345 nanoliters of ribonucleoprotein RDL mix followed by feeding with one molar sucrose before releasing the bees into the black experimental box. Feed a second set of eight bees as controls without any injections and release them into the white control box. Then, place both boxes into a polystyrene container with wet paper, observing the bees twice a day to ensure they have enough food and a good humidity.

48 hours after the injection, immobilize the bees on ice for 30 seconds and use scissors to decapitate each insect. Place the heads in 4%paraformaldehyde IN PBS under a dissecting microscope in a fume hood, and use barraquer iris scissors to carefully but rapidly remove the antennae, compound eyes, and the top exoskeleton. Allow the heads to sit in the fixative solution for 10 minutes before removing the rest of exo skeleton from the head and all of the remaining trachea.

Then, place each brain in a 1.5 milliliter microcentrifuge tube containing at least one milliliter of 4%paraformaldehyde overnight at four to eight degrees Celsius. The next morning, add 3.8 grams of argrose to 50 milliliters of distilled water in an erlenmeyer flask and microwave the solution until the argrose liquifies. Transfer three to four fixed honey bee brains into a new 35 millimeter petri dish and use tissue paper to remove the excess fixative.

Carefully pour the liquid argrose over the brains and orient the samples within the argrose so that the antennae lobes are facing up. After the argrose has solidified, use a scalpel to cut out individual blocks of argrose containing a single brain and fill each well of a 24-well plate loaded with one basket with a hydrophobic mesh bottom and 600 microliters of PBS per well. Then, use a vibratome to a acquire 70 micrometer cross sections of the brain tissue from each argrose block.

Placing the sections from a single brain onto the mesh of the same basket as they are acquired. After labeling the sections with fluorophore conjugated antibodies, according to standard protocols, embed each slide with sections from a single brain using a drop of mounting medium and visualize the samples by fluorescence microscopy. Anti-RDL labels neuropils in the frontal section of wild-type, but not CRISPR-Cas9 RDL knockout bee brains.

A similar specificity was observed for anti-mGlutR1 antibodies. In these representative, quantitative PCR drop off tests in bees brains injected with RDL CRIPSR-Cas9, the relative reduction of the fluoresence corresponded to the number of the modified guide DNA in the sample. After mGlutR1 CRISPR-Cas9 injection, the relative modification of guide DNA was approximately 59%in the brains of injected bees compared to the guide DNA observed in the brains of non-injected bees.

In this quantitative RT-PCR ina separate group of bees the relative reduction of the messenger RNA RDL was approximately 59%compared to the level of RNA in non-injected bees, and the relative reduction of the messenger mGlutR1 RNA in injected bees was approximately 53%Notably, the injection of RDL CRISPR-Cas9 through the ocelli might not always reach a large number of brain cells. For example, in these preparations only one be out of eight had RDL CRISPR-Cas9 in large numbers of brain cells compared to other bee brains. With the RDL CRISPR-Cas9 concentrated within the cells of the protocerebrum, but not the antennal lobe.

Be sure to avoid frozen and refrozen RNA. Take care not to overcool the bees. Harness them immediately once the stop moving in the vial.

This technique can be used to study the effects of the reduction of inhibitory ionotropic and metabotropic receptors in the mushroom body and central complex on honey bee behavior.