Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

Car T cell therapy is an innovative type of cancer immunotherapy that has led to remissions lasting over a decade. However, the rates of durable responses are still limited. With this protocol, we aim to enhance our understanding of the mechanisms behind resistance and to create tools to improve the response to CAR T cell therapy.

Many approaches are currently being employed to improve CAR T cell therapy, including combination therapies and optimization of CAR design. However, due to increasing evidence that the epigenetic profile of pre-infusion CAR T-cell products is an important determinative response, genetic editing technologies have become popular tools.

Genomic editing of CAR T cells has shown success in preventing toxicities and increasing the expansion and persistence of CAR T cells in preclinical and clinical studies. Our protocol for a genome-wide CRISPR knockout screen provides an efficient and unbiased approach to determine the best genetic edits to improve CAR T cell function.

Genome-wide gene perturbations require large-scale and adequate selection conditions to be effective. To study the development of CAR T cell exhaustion, we learned that we must stimulate over 80 million CAR T cells for two weeks to select optimal gene edits.

While our protocol was designed to evaluate the development of exhaustion, this protocol can be adjusted to evaluate the development of other mechanisms of resistance. By completing genome-wide screens in different settings, we may eventually determine the best edits to improve the overall responses to CAR T cell therapy.

[Narrator] After transducing the CAR T cells with the CRISPR library, centrifuge the samples at 300g for five minutes at 4 degrees Celsius to wash them. Aspirate the supernatant and resuspend the samples in 20 milliliters of T cell medium. To preserve samples for next-generation sequencing, aliquot enough CAR T cells into a 50-milliliter conical tube to maintain a guide RNA representation of 500 cells per guide RNA. Centrifuge the sample at 300g for five minutes at 4 degrees Celsius. Then resuspend the cell pellet in one milliliter of PBS and transfer it to a two-milliliter microcentrifuge tube. Wash the 50-milliliter conical tube with one milliliter of PBS and transfer the wash to the same microcentrifuge tube. Then centrifuge the samples at 500g for five minutes at 4 degrees Celsius. Remove the supernatant and store the cell pellets at -20 degrees Celsius. To start the co-culture, add CAR T cells to CD19-positive JeKo-1 cells at a one-to-one ratio. Bring the total volume of the co-culture to 160 milliliters with T cell medium and store the samples in an incubator at 37 degrees Celsius with 5% carbon dioxide. To re-stimulate the CAR T cells, collect the co-cultures into 50-milliliter conical tubes and centrifuge the samples at 300g for five minutes at 4 degrees Celsius. Resuspend each sample in 80 milliliters of T cell medium and add 80 times 10 to the power of six JeKo-1 cells in 80 milliliters of T cell medium. Store the samples in an incubator at 37 degrees Celsius with 5% carbon dioxide. On day 15, to isolate the CAR T cells from the co-culture, first count the cells. Combine a positive selection kit for CD4-positive T cells with a positive selection kit for CD8-positive T cells to isolate T cells from the CAR T JeKo-1 co-cultures. Then count the isolated T cells. To preserve the samples for next-generation sequencing, aliquot 33 times 10 to the power of six CAR T cells into a 50-milliliter conical tube and centrifuge the sample at 300g for five minutes at 4 degrees Celsius. Resuspend the cell pellet in one milliliter of PBS and transfer it to a two-milliliter microcentrifuge tube. Then wash the 50-milliliter conical tube with one milliliter of PBS and transfer the wash to the same microcentrifuge tube. After centrifuging the samples and removing the supernatant, store the cell pellets at -20 degrees Celsius. To begin, isolate genomic DNA from frozen CAR T cell pellets using a genomic DNA isolation kit. Purify the isolated genomic DNA through ethanol precipitation by first pre-chilling 100% ethanol to -20 degrees Celsius. Aliquot the eluted DNA equally into several microcentrifuge tubes, ensuring each aliquot is 200 to 250 microliters. Add two volumes of pre-chilled 100% ethanol, 0.1 volume of three molar sodium acetate, and one microliter of 20 milligrams per milliliter glycogen. Incubate the samples overnight at -20 degrees Celsius. On the second day, spin the samples at 13,000g for 20 minutes to pellet the precipitated DNA. Remove the supernatant and wash the pellets with one milliliter of 70% ethanol by spinning at 7,500g for 10 minutes. After completely drying the pellets, resuspend the pellets in 50 microliters of sterile water. To prepare the genomic DNA for next-generation sequencing, set up a PCR with the specified reaction mixture shown here. Then perform the PCR using the following cycling conditions. Pull the PCR reactions for each sample and purify the PCR product using a PCR purification kit according to the manufacturer's instructions. Run three micrograms of the PCR product for each sample on a 2% agarose gel. Remove the PCR product from the gel by extracting the DNA using a gel extraction kit according to the manufacturer's instructions. Store the extracted samples at -20 degrees Celsius.