Comparative Analysis of Cell Dilution Assays for Single-Cell Colony Formation in CRISPR-Mediated Knockout Screening

BACKGROUND/AIMS:The ribonucleic acid (RNA)-guided Cas9 nuclease from the microbial clustered regularly interspaced palindromic repeats (CRISPR) immune system enables genome editing in eukaryotic cells by using a 20-nucleotide target sequence guide RNA (gRNA).A key CRISPR technique is the ability to identify individual cells in 96-well plates, which highlights the major challenges of obtaining single-cell knockouts.Two distinct dilution strategies were applied to breast cancer cells-the standard dilution method and the two-gradual dilution method-to compare their efficiency in producing single-cell colonies. MATERIALS AND METHODS:It began with target design, CRISPR-mediated gene modifications, and single-cell assays.Following genetic modifications, successful cloning was confirmed by Sanger sequencing.MDA-MB-231 cells were transfected with the constructed Cas9 plasmid.Forty-eight hours after transfection, the cells were passaged for a single-cell colony assay.Cells were seeded in 96-well plates using two different methods.In the standard cell dilution method, 100 µL of medium was added to each well.A cell suspension containing 100 cells in 100 µL was placed in the first column, and serially diluted across the plate by stepwise transfer of 100 µL from one column to the next to the final column.In the two-gradual cell dilution method, 100 µl of medium was added to all wells except A1, where 2×104 cells in 200 µl were seeded.Cells were diluted down the A1-H1 column, and then 100 µl was transferred sequentially from column to column to the 12 th column, with the remaining volume discarded.Single-cell growth and colony formation were monitored by light microscopy at 24-hour intervals.RESULTS: Comparison of the two-gradual and standard cell dilution methods demonstrated that each plate in the two-gradual method yielded 8-9 single colonies, whereas the standard method yielded only 1-2 colonies per plate (p=0.0192).This result revealed that the two-step gradual dilution method offers a statistically significant advantage in obtaining a greater number of candidate knockout single cells compared with the standard approach.CONCLUSION: This methodological improvement substantially enhances the efficiency of genetic screening workflows and more robust experimental designs.Consequently, researchers are expected to achieve higher success rates in identifying and validating candidate knockout cells, thereby accelerating downstream functional studies and advancing the reliability of CRISPR-based applications.