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Article Open Access

Genome‑wide CRISPR screening identifies CLDN1 as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer

  • Authors:
    • Xiaogang Wang
    • Benzhong Mi
    • Jinfeng Lv
    • Xin Tang
    • Jingyu Huang
    • Fei Liu
    • Yajing Xing
    • Xingjian Wen
    • Yongde Wang
    • Yadong Zhong
  • View Affiliations / Copyright

    Affiliations: Chongqing Key Laboratory of Chinese Medicine and Health Science, Chongqing Academy of Chinese Materia Medica, Chongqing 400065, P.R. China, Institute for Silk and Related Biomaterials Research, Chongqing Academy of Animal Sciences, Chongqing 402460, P.R. China, School of Chinese Materia Medica, Chongqing University of Chinese Medicine, Chongqing 402760, P.R. China, School of Basic Medical Sciences, Chongqing University of Chinese Medicine, Chongqing 402760, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 62
    |
    Published online on: December 1, 2025
       https://doi.org/10.3892/ol.2025.15415
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Abstract

Berberine (BBR), a natural compound with diverse anticancer properties, exerts potent inhibitory effects on lung cancer cell proliferation. However, its particular molecular targets remain unknown. The present study aimed to identify the key genetic determinants that mediate the cellular response to BBR in lung cancer. To achieve this, genome‑wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 (Cas9) knockout screening was conducted in A549 cells, followed by functional validation and mechanistic assays. In the study, the genome‑wide CRISPR/Cas9 knockout screening in A549 lung cancer cells identified claudin‑1 (CLDN1) as a critical modulator of BBR sensitivity. Notably, whilst CLDN1 was enriched in the positive selection screen, its knockout markedly increased the sensitivity of A549 cells to BBR, thus leading to enhanced G1‑phase arrest and reduced proliferation. These findings suggest that CLDN1 could serve a dual role, promoting cellular resistance under selective pressure, while simultaneously demonstrating a therapeutic vulnerability when directly inhibited. Overall, the present study identified CLDN1 as a key mediator of the anticancer effects of BBR, thus providing a foundation for its potential development as a therapeutic target to optimize lung cancer therapy.
View Figures

Figure 1

Effect of BBR on A549 cell
proliferation. A549 cells were treated with different
concentrations of BBR (1, 2.5, 5, 10, 20, 40 and 60 µM) for 72 h,
and cell viability was assessed using a Cell Counting Kit-8 assay.
The data are expressed as the mean ± standard deviation. BBR,
berberine.

Figure 2

Genome-wide CRISPR/Cas9 screening in
A549 cells. (A) Schematic workflow of the CRISPR/Cas9 screening
strategy. (B) Western blot analysis of spCas9 protein levels in
A549 and P0 cells. CRISPR, clustered regularly interspaced short
palindromic repeats; Cas9, CRISPR-associated protein 9; spCas9,
Streptococcus pyogenes CRISPR-associated protein 9; sgRNA,
single-guide RNA; NGS, next-generation sequencing.

Figure 3

Enrichment analyses. (A) KEGG pathway
and (B) GO term enrichment analyses. (C) Top 10 differentially
expressed genes of positive and negative selection. KEGG, Kyoto
Encyclopedia of Genes and Genomes; GO, Gene Ontology.

Figure 4

Effect of CLDN1 knockout on
the response of A549 cells to BBR. (A) CRISPR/CRISPR-associated
protein 9-mediated CLDN1 knockout in A549 cells. (B) Loss of
CLDN1 protein expression assessed in A549-KO cells using
western blot analysis. (C) Cell viability of wild type A549 and
A549-KO cells treated with 2.5 µM BBR for 24, 48 and 72 h. Data are
expressed as the mean ± standard deviation (n=3). **P<0.01;
***P<0.001. (D) Western blot analysis of cyclin D1 expression in
A549 and A549-KO cells following treatment with BBR. (E) Flow
cytometry analysis of cell cycle distribution highlighting the
differences in the G1 and S phases between A549 and A549-KO cells.
BBR, berberine; CLDN1, claudin-1; CRISPR, clustered
regularly interspaced short palindromic repeats; A549-KO,
CLDN1-depleted A549 cells; KO, knockout.
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Copy and paste a formatted citation
Spandidos Publications style
Wang X, Mi B, Lv J, Tang X, Huang J, Liu F, Xing Y, Wen X, Wang Y, Zhong Y, Zhong Y, et al: Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer. Oncol Lett 31: 62, 2026.
APA
Wang, X., Mi, B., Lv, J., Tang, X., Huang, J., Liu, F. ... Zhong, Y. (2026). Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer. Oncology Letters, 31, 62. https://doi.org/10.3892/ol.2025.15415
MLA
Wang, X., Mi, B., Lv, J., Tang, X., Huang, J., Liu, F., Xing, Y., Wen, X., Wang, Y., Zhong, Y."Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer". Oncology Letters 31.2 (2026): 62.
Chicago
Wang, X., Mi, B., Lv, J., Tang, X., Huang, J., Liu, F., Xing, Y., Wen, X., Wang, Y., Zhong, Y."Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer". Oncology Letters 31, no. 2 (2026): 62. https://doi.org/10.3892/ol.2025.15415
Copy and paste a formatted citation
x
Spandidos Publications style
Wang X, Mi B, Lv J, Tang X, Huang J, Liu F, Xing Y, Wen X, Wang Y, Zhong Y, Zhong Y, et al: Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer. Oncol Lett 31: 62, 2026.
APA
Wang, X., Mi, B., Lv, J., Tang, X., Huang, J., Liu, F. ... Zhong, Y. (2026). Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer. Oncology Letters, 31, 62. https://doi.org/10.3892/ol.2025.15415
MLA
Wang, X., Mi, B., Lv, J., Tang, X., Huang, J., Liu, F., Xing, Y., Wen, X., Wang, Y., Zhong, Y."Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer". Oncology Letters 31.2 (2026): 62.
Chicago
Wang, X., Mi, B., Lv, J., Tang, X., Huang, J., Liu, F., Xing, Y., Wen, X., Wang, Y., Zhong, Y."Genome‑wide CRISPR screening identifies <em>CLDN1</em> as a central node in the anticancer mechanisms of berberine and as a therapeutic target in lung cancer". Oncology Letters 31, no. 2 (2026): 62. https://doi.org/10.3892/ol.2025.15415
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