CRISPR library screening for tumor drug resistance genes: How to solve the issue of low lentiviral packaging titer?

Low supernatant titer can be addressed by concentration. After collecting the supernatant, first centrifuge at 4°C to remove cell debris, filter through a 0.45 μm PES membrane, then concentrate by ultracentrifugation or PEG/ultrafiltration, which typically increases the titer by 1-2 orders of magnitude. Do not subject to repeated freeze-thaw cycles; aliquot and store at -80°C. During packaging, pretreatment with sodium butyrate or chloroquine in the culture medium can also increase yield. For library experiments, the key is to package a sufficient amount in a single batch to avoid compositional bias caused by multiple small-scale packaging batches. Functional titer should be determined by counting after drug selection, not merely by p24 or qPCR physical titer, which may overestimate.

If the lentiviral titer from continuous packaging of lentiCRISPR v2 remains low, consider switching to the more compact lentiGuide-Puro backbone combined with Cas9-stable cells, but this will change the experimental workflow. For serum, heat-inactivated FBS is recommended; 293T cells are sensitive to lot-to-lot variation, so test new serum for growth and transfection efficiency first. For consumables, scale up using 15 cm dishes or cell factories, and use low-protein-binding PES membranes for filtration. To save troubleshooting time, you can directly use commercial lentiviral packaging kits, which have fast lead times and already optimized formulations and plasmid ratios. Domestic fetal bovine serum and consumables generally arrive quickly, but inventory fluctuates greatly, so stock two batches in advance.

10^5 TU/mL is on the low side when unconcentrated but can be rescued. First check the plasmid ratios; typically the mass ratio of lentiCRISPR v2:psPAX2:pMD2.G is 4:3:1 or 3:1:1. When transfecting with PEI, the DNA:PEI mass ratio is about 1:3, but this varies between PEI lots, so a small-scale test is needed. Use low-passage 293T cells at 70%-80% confluence for transfection. When the library plasmid amount is large, mix it thoroughly in the transfection complex first and then add dropwise to avoid local overconcentration. Change the medium 6 h after transfection, collect the supernatant once each at 48 h and 72 h, combine them, and then concentrate. Do not collect only the 48 h time point.

For library screening, besides titer, library complexity must also be ensured. During transfection, the cell number should be at least 200–500 times the total sgRNA number; otherwise, even with a high titer, representativeness will be lost. For scale-up culture, use 15 cm dishes or cell factories. When the plasmid mixture volume is large, prepare PEI in multiple tubes to avoid local overconcentration. Set up a GFP control to monitor transfection efficiency; if it is below 60%–70%, re-optimization is required. Before drug selection, perform a kill curve to determine the puromycin concentration so that functional titers are comparable. Stock consumables in advance according to the scale-up size to avoid shortages midway that affect library quality.