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Cancer AND CRISPR-Cas9

Arthur R Gorter de Vries, Lucas G F Couwenberg, Marcel van den Broek, Pilar de la Torre Cortés, Jolanda Ter Horst, Jack T Pronk, Jean-Marc G Daran
Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome...
December 5, 2018: Nucleic Acids Research
Cheuk-Ting Law, Lai Wei, Felice Ho-Ching Tsang, Cerise Yuen-Ki Chan, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Daniel Wai-Hung Ho, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Chun-Ming Wong
Hepatocellular carcinoma (HCC) is the third lethal cancer worldwide. Increasing evidence showed that epigenetic alterations play an important role in human carcinogenesis. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, by systematically analyzing the expression of chromatin remodeling genes in human HCCs, we found that HELicase, Lymphoid-Specific (HELLS), a SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC...
December 5, 2018: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
Mi-Jin An, Dae-Hyun Kim, Chul-Hong Kim, Mijin Kim, Sangmyung Rhee, Sang-Beom Seo, Jung-Woong Kim
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most lethal cancer worldwide. Although gene mutations associated with HCC development have been intensively studied, how epigenetic factors specifically modulate the functional properties of HCC by regulating target gene expression is unclear. Here we demonstrated the overexpression of KDM3B in liver tissue of HCC patients using public RNA-seq data. Ablation of KDM3B by CRISPR/Cas9 retarded the cell cycle and proliferation of hepatocarcinoma HepG2 cells...
December 1, 2018: Biochemical and Biophysical Research Communications
Charles G Bailey, Cynthia Metierre, Yue Feng, Kinsha Baidya, Galina N Filippova, Dmitri I Loukinov, Victor V Lobanenkov, Crystal Semaan, John Ej Rasko
CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF , which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf ( Ctcf +/- ) mice...
November 30, 2018: International Journal of Molecular Sciences
Caigang Liu, Lisha Sun, Jie Yang, Tong Liu, Yongliang Yang, Se-Min Kim, Xunyan Ou, Yining Wang, Li Sun, Mone Zaidi, Maria I New, Tony Yuen, Qiyong Guo
Fibrous sheath interacting protein 1 (FSIP1) is a cancer antigen expressed in the majority of breast cancer tissues and is associated with poor prognosis. However, the role of FSIP1 in the progression and drug sensitivity of triple-negative breast cancer (TNBC) has not been explored. Here, we show that FSIP1 deficiency by shRNA-mediated knockdown or CRISPR-Cas9-mediated knockout significantly inhibits the proliferation and invasion of TNBC cells and impairs chemotherapy-induced growth inhibition in vivo. Computational modeling predicted that FSIP1 binds to ULK1, and this was established by coimmunoprecipitation...
December 3, 2018: Proceedings of the National Academy of Sciences of the United States of America
Ruilong Lan, Fei Huang, Guangxian Zhong, Ruiqing Chen, Zeng Wang, Junying Chen, Lengxi Fu, Jinsheng Hong, Lurong Hong
PURPOSE: Radioresistance is an important factor for unsatisfactory prognosis in Nasopharyngeal carcinoma (NPC) patients. Ubiquitous mitochondrial creatine kinase (CKMT1) is always associated with malignancy in a variety of cancers. However, its significance in NPC progression and radiosensitivity remains unclear.The present study focused on investigating the effects of CKMT1 on NPC cell radiosensitivity. MATERIALS AND METHODS: CKMT1 was overexpressed in NPC cell line CNE-1 or knocked out in CNE-2...
December 3, 2018: International Journal of Radiation Biology
Pawel Bialk, Yichen Wang, Kelly Banas, Eric B Kmiec
Recent studies point to the evolution of drug resistance in lung cancer as being centered, at least in part, on the upregulation of various genes involved in controlling efflux or drug inactivation. Among the most important of these genes is Nuclear Factor Erythroid 2-Related Factor ( NRF2 ), considered the master regulator of 100-200 target genes involved in cellular responses to oxidative and/or electrophilic stress. With increased focus on the development of combinatorial approaches for cancer treatment, we utilized CRISPR/Cas9 to disable the NRF2 gene in lung cancer cells by disrupting the NRF2 nuclear export signal (NES) domain; phenotypically, the protein is largely blocked from transiting into the nucleus after translation...
December 21, 2018: Molecular Therapy Oncolytics
Cai Liang, Zhenlei Zhang, Qinfu Chen, Haiyan Yan, Miao Zhang, Xingfeng Xiang, Qi Yi, Xuan Pan, Hankun Cheng, Fangwei Wang
The inner centromere region of a mitotic chromosome critically regulates sister chromatid cohesion and kinetochore-microtubule attachments. However, the molecular mechanism underlying inner centromere assembly remains elusive. Here, using CRISPR/Cas9-based gene editing in HeLa cells, we disrupted the interaction of Shugoshin 1 (Sgo1) with histone H2A phosphorylated on Thr-120 (H2ApT120) to selectively release Sgo1 from mitotic centromeres. Interestingly, cells expressing the H2ApT120-binding defective mutant of Sgo1 have an elevated rate of chromosome missegregation accompanied by weakened centromeric cohesion and decreased centromere accumulation of the chromosomal passenger complex (CPC), an integral part of the inner centromere and a key player in the correction of erroneous kinetochore-microtubule attachments...
November 29, 2018: Journal of Biological Chemistry
Lin Li, Weili Miao, Ming Huang, Preston Williams, Yinsheng Wang
Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC in humans. SET domain-containing 2 (SETD2), a lysine methyltransferase for histone and other proteins, has been found to be frequently lost in ccRCC. However, the mechanisms through which deficiency in SETD2 contributes to ccRCC development remain largely unknown. Here, we used a human embryonic kidney epithelial cell line with the SETD2 gene knocked out using CRISPR/Cas9 technology. Using ChIP-seq analysis, we showed that SETD2 loss leads to diminished occupancy of histone H3K36me3 and H4K16ac on actively transcribed genes...
November 28, 2018: Molecular & Cellular Proteomics: MCP
Tonći Šuštić, Sake van Wageningen, Evert Bosdriesz, Robert J D Reid, John Dittmar, Cor Lieftink, Roderick L Beijersbergen, Lodewyk F A Wessels, Rodney Rothstein, René Bernards
BACKGROUND: Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options. METHODS: We first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy...
November 27, 2018: Genome Medicine
Juanjuan Zhao, Quande Lin, Yongping Song, Delong Liu
Currently, the two approved T cell products with chimeric antigen receptors (CAR) are from autologous T cells. These CAR T cells approved for clinical use must be generated on a custom-made basis. This case-by-case autologous T cell production platform remains a significant limiting factor for large-scale clinical application due to the costly and lengthy production process. There is also an inherent risk of production failure. The individualized, custom-made autologous CAR T cell production process also posts constriction on the wide application on diverse tumor types...
November 27, 2018: Journal of Hematology & Oncology
Hui San Chin, Mark X Li, Iris K L Tan, Robert L Ninnis, Boris Reljic, Kristen Scicluna, Laura F Dagley, Jarrod J Sandow, Gemma L Kelly, Andre L Samson, Stephane Chappaz, Seong L Khaw, Catherine Chang, Andrew Morokoff, Kerstin Brinkmann, Andrew Webb, Colin Hockings, Cathrine M Hall, Andrew J Kueh, Michael T Ryan, Ruth M Kluck, Philippe Bouillet, Marco J Herold, Daniel H D Gray, David C S Huang, Mark F van Delft, Grant Dewson
Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function...
November 26, 2018: Nature Communications
Masayuki Fujii, Hans Clevers, Toshiro Sato
Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients' tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids...
November 23, 2018: Gastroenterology
Kensuke Otsuka, Masanori Tomita
Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated "Focicles" harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn)...
November 23, 2018: Scientific Reports
Sufang Zhang, Hsiao Hsiang Chao, Xiaoxiao Wang, Zhongtao Zhang, Ernest Y C Lee, Marietta Y W T Lee
Human DNA polymerase δ is normally present in unstressed, non-dividing cells as a heterotetramer (Pol δ4). Its smallest subunit, p12, is transiently degraded in response to UV damage, as well as during the entry into S-phase, resulting in the conversion of Pol δ4 to a trimer (Pol δ3). In order to further understand the specific cellular roles of these two forms of Pol δ, the gene (POLD4) encoding p12 was disrupted by CRISPR/Cas9 to produce p12 knockout (p12KO) cells. Thus, Pol δ4 is absent in p12KO cells, leaving Pol δ3 as the sole source of Pol δ activity...
November 13, 2018: DNA Repair
Gi-Cheon Kim, Ho-Keun Kwon, Choong-Gu Lee, Ravi Verma, Dipayan Rudra, Taemook Kim, Keunsoo Kang, Jong Hee Nam, Young Kim, Sin-Hyeog Im
Breast cancer is highly aggressive and is the leading cause of cancer-related mortality in women in developed countries. The ETS proto-oncogene 1 (Ets1) has versatile roles during the cellular processes of cancer development. It is often highly expressed in breast cancers and mediates migration and invasion of human breast cancer cells. However, underlying mechanisms of Ets1 gene expression is still ambiguous. Here, we identified a core-regulatory element (CRE) located in the Ets1 promoter region (-540/-80 bp from TSS) that contains elements responsible for associating with NFATs and NF-κBs...
November 23, 2018: Oncogenesis
Srikanth Barkeer, Seema Chugh, Saswati Karmakar, Garima Kaushik, Sanchita Rauth, Satyanarayana Rachagani, Surinder K Batra, Moorthy P Ponnusamy
BACKGROUND: Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs. METHODS: Effect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis...
November 22, 2018: BMC Cancer
Rosalie M Sterner, Reona Sakemura, Michelle J Cox, Nan Yang, Roman H Khadka, Cynthia L Forsman, Michael J Hansen, Fang Jin, Katayoun Ayasoufi, Mehrdad Hefazi, Kendall J Schick, Denise K Walters, Omar Ahmed, Dale Chappell, Tarek Sahmoud, Cameron Durrant, Wendy K Nevala, Mrinal M Patnaik, Larry Pease, Karen E Hedin, Neil E Kay, Aaron J Johnson, Saad S Kenderian
Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. While the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19 targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy...
November 21, 2018: Blood
Kizuki Yuza, Masato Nakajima, Masayuki Nagahashi, Junko Tsuchida, Yuki Hirose, Kohei Miura, Yosuke Tajima, Manabu Abe, Kenji Sakimura, Kazuaki Takabe, Toshifumi Wakai
BACKGROUND: Pancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. MATERIALS AND METHODS: S1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients...
December 2018: Journal of Surgical Research
Abhishek K Gupta, Tushar Murthy, Kiran V Paul, Oscar Ramirez, Joseph B Fisher, Sridhar Rao, Alexander B Rosenberg, Georg Seelig, Alex C Minella, Manoj M Pillai
Cancer-associated mutations of the core splicing factor 3 B1 (SF3B1) result in selection of novel 3' splice sites (3'SS), but precise molecular mechanisms of oncogenesis remain unclear. SF3B1 stabilizes the interaction between U2 snRNP and branch point (BP) on the pre-mRNA. It has hence been speculated that a change in BP selection is the basis for novel 3'SS selection. Direct quantitative determination of BP utilization is however technically challenging. To define BP utilization by SF3B1-mutant spliceosomes, we used an overexpression approach in human cells as well as a complementary strategy using isogenic murine embryonic stem cells with monoallelic K700E mutations constructed via CRISPR/Cas9-based genome editing and a dual vector homology-directed repair methodology...
November 20, 2018: Nucleic Acids Research
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