Related Experiment Video
Updated: Oct 4, 2025
Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells
Published on: May 30, 2025
Dual CRISPR interference and activation for targeted reactivation of X-linked endogenous FOXP3 in human breast cancer
Xuelian Cui1, Chao Zhang1, Zhifang Xu1
1Department of Genetics, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL, 35294, USA.
Background:
Unlike autosomal tumor suppressors, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers.
Methods:
Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins.
Results:
Using Streptococcus pyogenes dCas9-KRAB for silencing XIST and Staphylococcus aureus dCas9-VPR for activating FOXP3, we achieved CRISPR activation of FOXP3 in various cell lines of human female breast cancers. In human breast cancer HCC202 cells, which express a synonymous heterozygous mutation in the coding region of FOXP3, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 activation. Also, reactivation of endogenous FOXP3 in breast cancer cells by CRISPRi/a inhibited tumor growth in vitro and in vivo. We further optimized CRISPRa by fusing dCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and H3K4me3 and lower DNA methylation. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications.
Conclusions:
The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers.
Insights
Targeted reactivation of X-linked tumor suppressors, like FOXP3, via CRISPRi/a therapy shows promise for treating female breast cancers by reversing X-chromosome inactivation.
Area of Science:
- Genetics
- Epigenetics
- Cancer Biology
Background:
- X-linked tumor suppressors are susceptible to inactivation via a single mutation due to X-chromosome inactivation (XCI).
- Targeted reactivation of the non-mutated allele presents a potential therapeutic strategy for female breast cancers.
Discussion:
- A novel dual CRISPR interference and activation (CRISPRi/a) system was developed using orthogonal dCas9 proteins to simultaneously silence and reactivate X-linked genes.
- This CRISPRi/a approach achieved FOXP3 activation in human female breast cancer cells, with enhanced and prolonged activation observed when XIST was simultaneously silenced.
Key Insights:
- Reactivation of endogenous FOXP3 using CRISPRi/a demonstrated inhibition of tumor growth both in vitro and in vivo.
- Optimization of CRISPRa by fusing dCas9 to TET1 further enhanced FOXP3 activation.
- CRISPRi/a targeting altered epigenetic modifications, including DNA methylation and histone acetylation, at the XIST and FOXP3 loci.
Outlook:
- The simultaneous modulation of XIST and FOXP3 offers a valuable research tool.
- This approach holds potential as a therapeutic strategy for female breast cancers by reactivating silenced tumor suppressor genes.
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
02:31Homologous Recombination
01:58X-Inactivation
03:17Inheritance of Chromatin Structures