Dual CRISPR interference and activation for targeted reactivation of X-linked endogenous FOXP3 in human breast cancer cells

Xuelian Cui,Lei S Qi,Shuaibin Wang,Erica Stringer-Reasor,Lizhong Wang,Chao Zhang,Leiping Zeng,Sejong Bae,Dehua Zhao,Xin Li,Runhua Liu,Zhifang Xu

doi:10.1186/s12943-021-01472-x

Xuelian Cui, Lei S Qi + Show 10 more

Open Access

https://doi.org/10.1186/s12943-021-01472-x

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Abstract

BackgroundUnlike 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.MethodsTowards 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.ResultsUsing 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.ConclusionsThe 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.

Highlights

Autosomal tumor suppressor genes can be inactivated by a two-hit Knudson mechanism
For complex gene regulation, a flexible endonuclease-deficient CRISPR/Cas9-based platform that independently controls the expression of various genes within the same cell [2]
We developed, for human female breast cancer cells, a CRISPR interference (CRISPRi)/a approach for targeted transcriptional regulation of specific X-linked FOXP3, using two orthogonal deficient CRISPR/Cas9 (dCas9)-fusion systems, including SpdCas9-Krüppelassociated box (KRAB) for CRISPRi to the X-inactive specific transcript (XIST) locus and SadCas9-VP64-p65-Rta proteins (VPR) for CRISPR activation (CRISPRa) to the FOXP3 locus (Fig. S11)

Summary

Introduction

X chromosome-linked tumor suppressor genes, such as FOXP3 at Xp11.23 [1], can be inactivated by a single-hit mechanism, because of X-chromosome inactivation (XCI). For therapeutic purposes, targeted reactivation of XCIendogenous tumor suppressor genes may be an effective strategy to restore their function in female cancer cells. Using FOXP3 as an X-linked model gene, we aimed to develop, for human female breast cancer cells, a tunable and reversible, targeted reactivation of X-linked tumor suppressor genes. In the present study, using CRISPR interference and activation (CRISPRi/a), we achieved, for human female breast cancer cells, targeted reactivation of X-linked endogenous FOXP3, at least a partial reactivation from XCI. We argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers

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