In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

This protocol is significant because it allows researchers to simultaneously probe the tumor suppressive functions of hundreds of genes in head and neck cancer at a fraction of the cost of a conventional knockout mouse model. This technique is flexible and can be adapted to cover expression of genes to study their function in skin. The targeted CRISPR library protocol can also be applied for other cancer types.

Place the anesthetized animal ventral side up on the heated surgery stage and use surgical tape to hold the paws in place. Apply the nosecone anesthesia tube for a constant supply of isoflurane and oxygen until the surgery is completed. To confirm E9.5 pregnancy, apply a small quantity of hair removal cream to the abdomen and to spread it over a three-by-three centimeter squared area using a cotton tipped applicator.

After two to three minutes, gently remove the cream with gauze or tissue and wipe the area clean using PBS and 70%ethanol. Using the ultrasound probe and gel, check the pregnant mouse for embryonic day E9.5. This can also be done the day before at E8.5 to save time on the day of surgery.

Remove the ultrasound gel and wipe the area clean using BPS and then 70%ethanol. Inject the most with subcutaneous analgesic. Then you sterile forceps and scissors to make a vertical incision in the skin.

Use blunt forceps to separate the skin around the incision from the peritoneum. Then you sterile forceps and scissors to cut through the peritoneum. Using blunt forceps, gently pull out the left and right uterus horn and count the embryos.

Reinsert most of the uterine horns, but leave the distal end of one uterine horn containing three embryos exposed. Using blunt forceps, gently push and pull the uterus with the three embryos, through the opening in the silicone membrane of the modified Petri dish. Stabilize the Petri dish using cubes of modeling clay.

Stabilize the uterus with the three embryos inside the Petri dish, using a silicone mold. Then flatten the silicone plug at the side of the embryos using a blunt tool. Fill the Petri dish with sterile PBS and flush the silicone membrane on the bottom of the Petri dish, with the pregnant dam’s belly, thereby preventing any leakage.

Move the ultrasound head into the Petri dish, approximately 0.5 centimeters above the top embryo and adjust the stage so that the amniotic cavity becomes clearly visible in the ultrasound view. Align the injector needle, carefully into the modified Petri dish. Using the micromanipulator, place the tip of the needle within five millimeters of the top embryo, then toggle the needle back and forth to move into the plane where its tip appears the brightest in the ultrasound image.

Using the micromanipulator, push the needle through the uterine wall into the amniotic cavity. Inject 62 nanoliters of the lentivirus with the sgRNA library at a slow injection speed. Press the Inject button eight times for a total of 496 nanoliters per embryo.

Repeat the same procedure for the other two embryos. Then lift the ultrasound head and remove the silicone plug. Gently pushed the first two of the three embryos into the mouse abdomen using a sterile cotton swab.

Pull out the next three embryos and push in the previously injected third embryo. Repeat the injection procedure until the desired number of embryos are injected, making sure to not exceed 30 minutes of anesthesia. When finished, lift the ultrasound head, remove the micromanipulator with the needle, aspirate the PBS and remove the Petri dish.

Using sterile wipes, absorb any PBS that might have accumulated in the abdominal cavity. Close the peritoneal incision using absorbable sutures. Then use two staples to close the incision in the abdominal skin.

Next, generation sequencing reads of PCR amplified DNA from plasmid and lentiviral library transduced cells should show a high correlation. If the sgRNA reads from plasmid DNA do not show equal representation of sgRNAs, then the viral preparation and concentration procedure must be repeated with care. The results of the ultrasound-guided injection of lentivirus carrying Cre recombinase in E9.5 Lox-STOP-Lox confetti mouse pups at postnatal day P0, are shown here.

While high titer of virus would result in larger coverage of the mouse’s skin, it would also result in transduction of multiple sgRNAs into the same cell. This plot shows next-generation sequencing reads of PCR-amplified DNA, from plasmid and lentiviral library transduced cells, as well as reads from four representative tumors. sgRNA guides targeting tumor suppressors ADAM10 and RIPK4 are enriched in the tumor samples compared to the plasmid pool or infected cells.

Performing the surgery at E9.5 is critical. Remember to check for pregnancy with ultrasound, nine days after setting up the cross and identify the embryonic time point in each female mouse. Once the pups are born, the transduced cells can be isolated and extensively profiled using the latest gene profiling techniques such as single-cell RNA sequencing.