Table 7.

Troubleshooting CRISPR-suppressor scanning experiments

Problem	Possible Cause	Solution
Insufficient number of colonies is observed following library cloning transformation.	Gibson Assembly reaction was low efficiency. Transformed DNA is low quality. Electroporation was low efficiency. Insufficient cells were electroporated.	Further purify sgRNA inserts and/or digested lentiCRISPRv2 vector using a PCR purification kit to remove any contaminates left over from gel purification. Ensure no residual ethanol from the wash is present when resuspending sample at the end of the isopropanol precipitation. Adjust electroporation conditions used to ensure that cuvette does not arc, and minimize lag time between electroporation and resuspension of the cells in recovery medium. Scale up the number of cells electroporated by performing additional electroporations.
Sequenced sgRNA library shows poor statistics (e.g., many undetected sgRNAs, high skew ratio).	Errors or biases were introduced during PCR amplification steps.	Optimize cycle numbers for rounds 1 and 2 of PCR amplification for sgRNA library cloning, ensuring that less than 35–40 cycles are used in total across both amplifications. Optimize cycle numbers used in library amplification for NGS analysis, testing cycle numbers less than the recommended 22 cycles. Ensure that a high-fidelity DNA polymerase is used, such as Q5 hot-start high fidelity DNA polymerase.
Low lentiviral titer is observed.	Poor quality HEK293T cells were used for lentivirus production. Poor quality DNA was used for transfection. Non-optimal transfection conditions were used. Virus was handled improperly. Non-optimal transduction conditions were used. Cell line of interest is difficult to transduce.	Ensure that HEK293T cells are low passage (passage <10) and do not exceed 70–80% confluency at any time prior to seeding for transfection. Verify the quality of pMD2.G and psPAX2 plasmids by sequencing or diagnostic digest and repurify if necessary. For transfection, ensure that FuGENE HD is pipetted directly into the liquid in the tube and not against the tube walls, and limit incubation to 10–15 min. Optimize the volume of FuGENE HD used for transfection relative to the total amount of DNA. Ensure that lentivirus is snap-frozen rapidly on dry ice and is not subjected to additional freeze-thaw cycles. Thaw lentivirus slowly on ice. Concentrate lentivirus upon harvesting and prior to freezing, especially if the cell line of interest is difficult to transduce. Optimize transduction conditions by adjusting the number of cells seeded per well, the volume of lentivirus, and the polybrene concentration. Optimal conditions will vary for each cell line. Scale up the number of cells transduced to achieve coverage.
Number of cells transduced during the screen is unexpectedly high or low.	Cells were handled inconsistently between titering transduction and screen transduction. Lentivirus aliquots became degraded.	Ensure that cells are handled the same way, and that transductions are performed using identical conditions, between titering and screening experiments. Calculate the number and proportion of cells successfully transduced. If these metrics are within acceptable MOI and coverage recommendations, proceed with the screen. Ensure that lentivirus used for titering is treated the same way as that used for the final screen (i.e., subjected to a freeze-thaw cycle, thawed on ice, etc.). Avoid subjecting lentivirus aliquots to multiple freeze-thaw cycles.
Low viability of cells is observed following drug selection during CRISPR-suppressor scanning experiment.	Too high of a drug concentration was used.	Reduce the concentration of drug used (i.e., use a less stringent selection).
Analysis of CRISPR-suppressor scanning results shows high replicate variability or unexpected behavior of controls.	Adequate coverage of the sgRNA library was not achieved. For example, cell populations were bottlenecked during the screen or an insufficient amount of DNA was harvested and amplified.	Optimize transduction conditions and carefully monitor cell counts during the screen to ensure that adequate coverage (ideally 1,000× coverage of the library) is maintained at all times. Scale up the number of cells subjected to drug treatment or reduce the concentration of drug used. Harvest an excess number of cells to ensure sufficient DNA is extracted.