Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells

OPEN ARTICLE Citation: Cell Discovery (2017) 3, 17034; doi:10.1038/celldisc.2017.34 www.nature.com/celldisc Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells Hyeung Kim1, Feng Li2, Quanyuan He1, Tingting Deng2, Jun Xu3, Feng Jin4, Cristian Coarfa4, Nagireddy Putluri4, Dan Liu1,3, Zhou Songyang1,2,* 1 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; 2Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China; 3Cell-Based Assay Screening Service Core, Baylor College of Medicine, Houston, TX, USA; 4Department of Molecular and Cellular Biology and Advanced Technology Core, Baylor College of Medicine, Houston, TX, USA CRISPR/Cas9 technology enables efficient loss-of-function analysis of human genes using somatic cells. Studies of essential genes, however, require conditional knockout (KO) cells. Here, we describe the generation of inducible CRISPR KO human cell lines for the subunits of the telosome/shelterin complex, TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, which directly interact with telomeres or can bind to telomeres through association with other subunits. Homozygous inactivation of several subunits is lethal in mice, and most loss-of-function studies of human telomere regulators have relied on RNA interference-mediated gene knockdown, which suffers its own limitations. Our inducible CRISPR approach has allowed us to more expediently obtain large numbers of KO cells in which essential telomere regulators have been inactivated for biochemical and molecular studies. Our systematic analysis revealed functional differences between human and mouse telomeric proteins in DNA damage responses, telomere length and metabolic control, providing new insights into how human telomeres are maintained. Keywords: CRISPR/Cas9; inducible knockout; metabolism; POT1 isoform; telomere; telosome/shelterin Cell Discovery (2017) 3, 17034; doi:10.1038/celldisc.2017.34; published online 26 September 2017 Introduction In the past 20 years, we have gained tremendous insight into how the ends of mammalian chromosomes or telomeres are maintained and regulated. Together with the telomerase, which consists of the reverse transcriptase TERT and RNA template TR/TERC, a multitude of telomere-binding proteins participate in telomere maintenance [1–5]. In particular, six core telomeric proteins, TRF1, TRF2, RAP1, TPP1, TIN2 and POT1, dynamically assemble on telomeres as a large complex called telosome or shelterin and are essential in telomere length regulation and end protection in mammals [6–8]. Extensive research has revealed the interactions and functions of telosome components. For *Correspondence: Zhou Songyang Tel: +713 798 5220; Fax: +713 796 9438 E-mail: Received 15 March 2017; accepted 27 July 2017 instance, TRF1 and TRF2 bind directly to the telomere duplex through their myb domains [9–13], whereas POT1 binds 3’ single-stranded (ss) telomeric overhangs [14, 15]. RAP1 is recruited by TRF2, but apparently does not directly interact with any of the other subunits [16]. TIN2 can interact with both TRF1 and TRF2 [6, 17–21]. It also binds TPP1 and helps bring to telomeres the TPP1-POT1 heterodimer that is essential for regulating telomerase access to telomeres [21–30]. The core telomere proteins often act as interaction hubs to recruit factors of diverse pathways to telomeres and ensure crosstalk between telomere maintenance pathways and other cellular processes [8, 19, 31, 32]. In fact, several key telomere regulators have been shown to regulate metabolism, providing direct evidence of the close ties between telomere regulation and metabolic control. For example, the human telomerase reverse transcriptase has been found to localize to the mitochondria and reduce intracellular oxidative stress [33–36]. Our lab has found Studying telosome subunits using inducible knockout cell lines 2 that TIN2 can also localize to the mitochondria and regulate oxidative phosphorylation [37]. Numerous studies have demonstrated that dysfunctional telomeres can lead to telomere length defects, deprotected telomeres, genomic instability and diseases [1, 4, 32, 38]. Much of our knowledge regarding the molecular and functional significance of mammalian telomeric proteins comes from studies using mouse knockout (KO) mouse embryonic fibroblast (MEF) cells, as genes are more readily targeted in mouse embryonic stem cells. However, notable differences exist in telomere regulation between mouse and human. For instance, human telomeres are considerably shorter than those of laboratory mice and human has one POT1 gene, whereas mouse has two (Pot1a and Pot1b). Such disparities underscore the need for loss-of-function human cellular models. Majority of the loss-of-function studies in human cells have relied on RNA interference (RNAi)-mediated inhibition of endogenous genes. The limitations of RNAi knockdown (KD) and the fact that several key telomere regulators including TRF2 and TIN2 are essential genes have complicated data analysis and interpretation. Complete inactivation of these telomere regulatory genes in cells may cause cell death, precluding further detailed biochemical and molecular studies, especially experiments that require extended culturing and/or large numbers of cells. The advent of the CRISPR/Cas9 genome-editing technology has afforded investigators unprecedented opportunities to more efficiently and specifically target genes in human cells and to explore the consequences of their inactivation [39–47]. In this study, we took advantage of the highly flexible and adaptable CRISPR/Cas9 system and generated human inducible KO cell lines for each of the telosome components. This panel of cells has allowed us to survey the functional significance of each telomeric protein and probe the impact of individual subunit inhibition on telomere regulation as well as metabolic control. With this systematic analysis of the function of human telomere proteins using inducible KO cell lines, we are able to better delineate the differences between mouse and human telomere biology. In addition, our panel of inducible KO cell lines should prove invaluable to investigators seeking to further explore the consequences of telomere dysfunction and to study how diverse cellular functions may be disrupted upon telomere dysregulation. Results Using CRISPR/Cas9 to generate inducible KO human cell lines Trf1, Trf2 and Tin2 have been reported to be essential genes in mouse [48–50]. To determine the roles of their human orthologs, we first turned to RNAi KD in human cells through stable expression of short hairpin RNAs (Supplementary Figure S1A). Even with effective KD (480%) of TRF2, for example, we could only observe minor DNA damage responses (DDRs) at telomeres (data not shown), rarely more severe p (...truncated)