CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells.

Luan Wen,Peter X Ma,Jun Song,Changzhi Zhao,Jinxue Ruan,Linyuan Ma,Jifeng Zhang,Jie Xu,Y Eugene Chen,Xiaofeng Xia

doi:10.3390/ijms21020653

Abstract

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.

Highlights

Telomeres are the unique structures in eukaryotic cells that maintain chromosome integrity
TERC is constitutively expressed in all cells examined; whereas TERT is expressed in cancer cells (Hela, PANC1 and SUM15) and human induced pluripotent stem cells, but not in primary cells such as human aortic smooth muscle cells (HASMCs) (Supplementary Figure S1A)
The relative telomere length/content normalized to single copy gene, referred to as the T/S ratio [27], measured by quantitative PCR is highest in the induced pluripotent stem cells (iPSCs), moderate in HASMCs, but extremely low in all cancer cells (Supplementary Figure S1B)

Summary

Introduction

Telomeres are the unique structures in eukaryotic cells that maintain chromosome integrity. Vertebrates telomeres consist of tandem repeats of 5 -TTAGGG-3 sequences. Shelterin proteins including TRF1, TRF2, POT1, TIN2, TPP1, and RAP1 bind telomeres to protect chromosome ends [1]. In most somatic cells where telomere maintenance mechanisms (TMM) are silent, telomeres are shortened at each cellular division due to the end replication problem, progressively leading to replicative senescence [2]. In stem cells and cancer cells, TMM is activated to overcome telomeric DNA attrition and replicative senescence [3]. There are two major TMM pathways: the primary one is telomerase-dependent, and the other is the telomerase-independent alternative lengthening of telomeres (ALT)

Methods

Results

Conclusion