Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes

This research focuses on genome-wide CRISPR screen and radiotherapy. We provide a protocol to view radio-sensitive and radio-resistant genes using a genome-wide CRISPR screen in lung cancer cell after irradiation. The current experimental challenges include the off-target effects, which is caused by the vast complexity of genome and potential difficulties in exploring the underlying mechanisms. Compared with traditional screen methods, CRISPR achieves permanent genetic modification and shows superior precision, which makes it especially valuable in functional genomic research and target discovery. In the future, our team will focus on studying in vivo CRISPR screen to solve the problems that this research left behind, and we will be committed to optimizing CRISPR technology.

[Narrator] To begin, adjust the adherent cell density to five times 10 to the power of five cells per milliliter. Using a pipette, distribute two milliliters of the cell suspension into each 3.5 centimeter culture dish for radiation treatment at different doses. Place the dishes in an incubator set to 37 degrees Celsius with 5% carbon dioxide and incubate overnight. Number each 3.5 centimeter culture dish from one to five, using a marker. Using a radiation source, administer radiation doses of 2, 4, 6, and 8 gray, respectively, to dishes two through five. Adjust the irradiated cell density to one times 10 to the power of five cells per milliliter. Seed 10 microliters per well, corresponding to 1000 cells per 100 microliters into six-well plates with three replicates per radiation dose. Then seed 30 microliters per well, corresponding to 3000 cells per 100 microliters into 96-well plates with five replicates per radiation dose. Now, mix the CCK-8 reagent with RPMI 1640 medium without FBS in a one to nine ratio. Add the mixture to the 96-well plate and incubate the plate in the dark for one hour. Then use a microplate reader to measure the optical density at 450 nanometers. To begin the infection process, set up a logarithmic concentration gradient for lentivirus from zero to 800 units per milliliter. Add the corresponding volume of lentivirus to two microliters of polybrene per dish and let it equilibrate at room temperature for five minutes. Slowly drip the lentivirus polybrene mixture into each well. Adjust the parental cell density to three times 10 to the power of five cells per milliliter and inoculate one milliliter into each well of a 12-well plate. Add puromycin in a concentration gradient to the wells. After 72 hours of infection, replace the medium in each well with complete medium containing the minimum puromycin concentration for cell killing. Calculate the multiplicity of infection for each well based on surviving cells. Adjust the adherent cell density to one times 10 to the power of seven cells per milliliter. Add lentivirus at a multiplicity of infection equal to 0.3 into 30 microliters per dish of polybrene and allow it to equilibrate at room temperature for five minutes. Slowly drip the lentivirus and polybrene mixture into the 15 centimeter culture dish. Mix well and incubate it overnight at 37 degrees Celsius with 5% carbon dioxide. On the second day post infection, aspirate the medium from the culture dish and replace it with 15 milliliters of RPMI 1640 complete medium containing 10% FBS. Repeat the same treatment for the uninfected parental cells as a negative control and continue culturing for 72 hours. Now, digest the cells from one 15 centimeter culture dish using 0.25% trypsin. Resuspend the cells in RPMI 1640 complete medium with 10% PBS and count the number of cells. After extracting the genomic DNA for day zero, use a NanoDrop UV spectrophotometer to measure DNA concentration and purity. Administer an appropriate dose of radiation to cells in the treatment group and leave control group cells untreated to propagate normally. After 14 days of treatment, digest both the treatment and control group cells using 0.25% trypsin. Resuspend the cells in RPMI 1640 complete medium with 10% FBS. Centrifuge the cells at 300 G for five minutes and discard the supernatant. Resuspend the pellet in one milliliter of PBS. After repeating the centrifugation step, extract day 14 genomic DNA from the pellet and determine the DNA concentration. Next, prepare the required primers and dilute them to 10 micromolar. After adding the components to set up a 20 microliter reaction system, centrifuge the tube briefly at 300 G for five seconds. For agarose gel electrophoresis, prepare the gel, remove the comb from it, and fill the electrophoresis tank with sufficient buffer to cover the gel. Add loading buffer to the DNA sample and mix well. Finally, load the mixture into the wells and start the electrophoresis Colony formation after 14 days revealed that exposure to two Gray of radiation significantly reduced the number of surviving colonies compared to zero Gray. The CCKA assay showed a substantial decline in cell viability at two Gray with further decrement at higher radiation doses. Treatment with increasing concentrations of puromycin for 72 hours showed that one micromolar was the minimum concentration required to eliminate A549 cells. PCR validation showed distinct bands at 231 base pairs, confirming the expected length of sgRNA sequences in the CRISPR library. Sequencing analysis revealed that approximately 60% of the reads successfully mapped to the reference genome. sgRNA read counts followed a Poisson distribution, matching theoretical expectations for a genome-scale screen. PCA and heat map analysis showed high intergroup variability and low intergroup variation, validating experimental consistency. Gene ontology analysis identified DNA damage response as a top enriched pathway among the top 15 results.