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10.1186/s13059-015-0791-1 http://scihub22266oqcxt.onion/10.1186/s13059-015-0791-1 C4640169!4640169 !26553065     free     free     free Warning: file_get_contents(https://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=26553065 &cmd=llinks): Failed to open stream: HTTP request failed! 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A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression |pdf=|usr=}}{{26553065 }} gab.com Text A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression JO: Genome Biol http://www.kidney.de/pget.php?&tw=1&pmid=26553065 #FOAvip BACKGROUND: To facilitate indefinite proliferation, stem cells and most cancer cells require the activity of telomerase, which counteracts the successive shortening of telomeres caused by incomplete DNA replication at the very end of each chromosome. Human telomerase activity is often determined by the expression level of telomerase reverse transcriptase (TERT), the catalytic subunit of the ribonucleoprotein complex. The low expression level of TERT and the lack of adequate antibodies have made it difficult to study telomerase-related processes in human cells. RESULTS: To overcome the low CRISPR-Cas9 editing efficiency at the TERT locus, we develop a two-step "pop-in/pop-out" strategy to enrich cells that underwent homologous recombination (HR). Using this technique, we fuse an N-terminal FLAG-SNAP-tag to TERT, which allows us to reliably detect TERT in western blots, immunopurify it for biochemical analysis, and determine its subcellular localization by fluorescence microscopy. TERT co-localizes detectably with only 5-7 % of the telomeres at a time in S-phase HeLa cells; no nucleolar localization is detected. Furthermore, we extend this approach to perform single base-pair modifications in the TERT promoter; reverting a recurrent cancer-associated TERT promoter mutation in a urothelial cancer cell line results in decreased telomerase activity, indicating the mutation is causal for telomerase reactivation. CONCLUSIONS: We develop a two-step CRISPR-Cas9 genome editing strategy to introduce precise modifications at the endogenous TERT locus in human cell lines. This method provides a useful tool for studying telomerase biology, and suggests a general approach to edit loci with low targeting efficiency and to purify and visualize low abundance proteins. Twit Text FOAVip A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression ????Genome Biol http://www.kidney.de/pget.php?&tw=1&pmid=26553065 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=26553065 #FOAvip English Wikipedia <ref>{{Cite pmid|26553065 }}</ref> A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression #MMPMID26553065 Xi L ; Schmidt JC ; Zaug AJ ; Ascarrunz DR ; Cech TR Genome Biol 2015[Nov]; 16 (ä): 231 PMID26553065 show ga BACKGROUND: To facilitate indefinite proliferation, stem cells and most cancer cells require the activity of telomerase, which counteracts the successive shortening of telomeres caused by incomplete DNA replication at the very end of each chromosome. Human telomerase activity is often determined by the expression level of telomerase reverse transcriptase (TERT), the catalytic subunit of the ribonucleoprotein complex. The low expression level of TERT and the lack of adequate antibodies have made it difficult to study telomerase-related processes in human cells. RESULTS: To overcome the low CRISPR-Cas9 editing efficiency at the TERT locus, we develop a two-step "pop-in/pop-out" strategy to enrich cells that underwent homologous recombination (HR). Using this technique, we fuse an N-terminal FLAG-SNAP-tag to TERT, which allows us to reliably detect TERT in western blots, immunopurify it for biochemical analysis, and determine its subcellular localization by fluorescence microscopy. TERT co-localizes detectably with only 5-7 % of the telomeres at a time in S-phase HeLa cells; no nucleolar localization is detected. Furthermore, we extend this approach to perform single base-pair modifications in the TERT promoter; reverting a recurrent cancer-associated TERT promoter mutation in a urothelial cancer cell line results in decreased telomerase activity, indicating the mutation is causal for telomerase reactivation. CONCLUSIONS: We develop a two-step CRISPR-Cas9 genome editing strategy to introduce precise modifications at the endogenous TERT locus in human cell lines. This method provides a useful tool for studying telomerase biology, and suggests a general approach to edit loci with low targeting efficiency and to purify and visualize low abundance proteins. |CRISPR-Cas Systems/*genetics [MESH] |Chromosomes/genetics [MESH] |DNA Replication/genetics [MESH] |Gene Expression Regulation [MESH] |Genome, Human [MESH] |HeLa Cells [MESH] |Humans [MESH] |Mutation [MESH] |Promoter Regions, Genetic [MESH] |RNA Editing/*genetics [MESH] |Telomerase/biosynthesis/*genetics [MESH]A novel two-step genome editing strategy with CRISPR-Cas9 provides new(epmc).pdf DeepDyve Pubget Overpricing