CRISPR/Cas9 library screening technology enables high-throughput functional genomics on a genome-wide scale. The process involves designing 3-10 sgRNAs per gene, synthesized via microchip technology as a pool of tens of thousands of probes covering the entire genome.

These probes are cloned into vectors to create a library, which is then packaged into lentiviruses. Host cells are infected at a low Multiplicity of Infection (MOI) to ensure that, theoretically, each cell integrates only a single sgRNA, resulting in a comprehensive pool of gene-knockout cells. Following phenotypic selection, genomic DNA is extracted, and the integrated sgRNA sequences are analyzed via Next-Generation Sequencing (NGS). By comparing the abundance of sgRNAs before and after selection, candidate genes associated with the phenotype can be identified.

In a CRISPR screening workflow, the ideal scenario is one where external variables—aside from the specific selection pressure (e.g., drug treatment)—are minimized. Consequently, achieving high viral titers, high purity, and a contamination-free environment is critical.

To assist in your research, we have compiled the following troubleshooting guide addressing common challenges in lentiviral packaging and transduction during CRISPR library screening.

Cause 1: Excessive cell plating density
Solution: Seed cells at a density of 4-5 × 10⁶ cells per 10 cm dish. If the cell line has a rapid doubling time, reduce the initial seeding density accordingly. Ensure cells are at 50-80% confluence at the time of transduction.

Cause 2: Premature assessment of transfection efficiency
Solution: Transgene expression typically reaches its peak approximately 48 hours post-transduction. Efficiency should be evaluated only after this incubation period.

Cause 3: Low viral titer
Solution: Concentrate the viral supernatant (up to 100-fold) using non-ultracentrifugation methods.

Cause 4: Poor cell viability during transfection
Solution:
(1) The viral packaging medium may contain components detrimental to cell growth; consider diluting the viral supernatant or shortening the incubation time.
(2) Optimize Polybrene usage: Determine the minimum effective concentration through titration or reduce the total exposure time.
(3) Ensure cells are in the logarithmic growth phase and in optimal health prior to transfection.

Cause 1: Premature viral harvesting
Solution: Harvest the virus at 48-72 hours post-transfection.

Cause 2: Absence or suboptimal concentration of Polybrene
Solution: Add 4 μg/ml Polybrene during transduction or optimize the concentration within the range of 2-12 μg/ml.

Cause 3: Multiple freeze-thaw cycles
Solution: Each freeze-thaw cycle reduces the titer by 2-4 fold; therefore, minimize the number of freeze-thaw cycles.

Cause 4: Suboptimal selection concentration used during titer measurement
Solution: Establish a kill curve for the specific cell line prior to titration to determine the optimal selection concentration.

Cause 1: Excessive MOI
Solution: Determine the optimal viral dosage via titration and dilute the virus accordingly.

Cause 2: Polybrene-induced toxicity
Solution: Reduce or optimize the concentration of Polybrene; shorten the incubation time during infection.

Cause 3: Toxicity from the viral supernatant or packaging medium
Solution: Use fresh culture medium to dilute the virus and during the harvesting process.

Cause: Cell sensitivity to Cas9 protein expression
Solution: Titrate the Cas9 virus using the target cells to ensure a single-copy integration (one virus per cell), or switch to an alternative cell line that is not sensitive to Cas9.