Arnab Datta, Chin-Rang Yang, Kavee Limbutara, Chung-Lin Chou, Markus M Rinschen, Viswanathan Raghuram, Mark A Knepper
Vasopressin regulates renal water excretion by binding to a Gα s-coupled receptor (V2R) in collecting duct cells, resulting in increased water permeability through regulation of the aquaporin-2 (AQP2) water channel. This action is widely accepted to be associated with cAMP-mediated activation of protein kinase A (PKA). Here, we use phosphoproteomics in collecting duct cells in which PKA has been deleted (CRISPR-Cas9) to identify PKA-independent responses to vasopressin. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent...
March 26, 2020: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Liwei Lang, Yong Teng
Transgene-based reporter gene assays have been used for discovery of inhibitors targeting vital gene transcription. In traditional assays, the reporter gene is commonly fused with a cloned promoter and integrated into a random genomic location. This has been widely applied but significantly dampened by disadvantages, including incomplete cis-acting elements, the influence of foreign epigenetic environments, and generation of false hits that disrupt the luciferase reporter activity. Therefore, there is a need to develop novel strategies for developing in situ reporter assays closely mimicking endogenous gene expression without disrupting its function...
2020: Methods in Molecular Biology
Jenq-Kuen Huang, Kadidia Samassekou, Hekmat B Alhmadi, David R VanDerway, Joshua D Diaz, Jacob A Seiver, Shawn W McClenahan, Scott M Holt, Lisa Wen
Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid...
2020: PloS One
Caroline Mahendra, Kathleen A Christie, Beatriz A Osuna, Rafael Pinilla-Redondo, Benjamin P Kleinstiver, Joseph Bondy-Denomy
CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19...
April 2020: Nature Microbiology
Veronika Mancikova, Helena Peschelova, Veronika Kozlova, Aneta Ledererova, Adriana Ladungova, Jan Verner, Tomas Loja, Frantisek Folber, Jiri Mayer, Sarka Pospisilova, Michal Smida
BACKGROUND: While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy. METHODS: First, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable ( IGHV )-unmutated (9 ATM -mutated, 8 TP53 -mutated, and 9 without mutations in ATM , TP53 , NOTCH1 or SF3B1 ) and 6 IGHV -mutated samples without mutations in the above-mentioned genes...
March 2020: Journal for Immunotherapy of Cancer
Russell T Walton, Kathleen A Christie, Madelynn N Whittaker, Benjamin P Kleinstiver
Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG capable of targeting an expanded set of NGN PAMs, and further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN>NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs...
March 26, 2020: Science
Taylor K Loe, Julia Su Zhou Li, Yuxiang Zhang, Benura Azeroglu, Michael Nicholas Boddy, Eros Lazzerini Denchi
Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur...
March 26, 2020: Genes & Development
Helen H N Yan, Hoi Cheong Siu, Siu Lun Ho, Sarah S K Yue, Yang Gao, Wai Yin Tsui, Dessy Chan, April S Chan, Jason W H Wong, Alice H Y Man, Bernard C H Lee, Annie S Y Chan, Anthony K W Chan, Ho Sang Hui, Arthur K L Cheung, Wai Lun Law, Oswens S H Lo, Siu Tsan Yuen, Hans Clevers, Suet Yi Leung
OBJECTIVE: Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. DESIGN: We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. RESULTS: We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions...
March 26, 2020: Gut
Xu-Hua Mo, Hui Zhang, Tian-Min Wang, Chong Zhang, Cong Zhang, Xin-Hui Xing, Song Yang
The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9)...
March 25, 2020: Applied Microbiology and Biotechnology
Dianhui Wu, Wenjuan Xie, Xiaomin Li, Guolin Cai, Jian Lu, Guangfa Xie
Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3 -c...
March 25, 2020: Applied Microbiology and Biotechnology
Shiwu Li, Yifan Li, Xiaoyan Li, Jiewei Liu, Yongxia Huo, Junyang Wang, Zhongchun Liu, Ming Li, Xiong-Jian Luo
Major depressive disorder (MDD) is one of the most prevalent psychiatric disorders and a leading cause of disability worldwide. Though recent genome-wide association studies (GWAS) have identified multiple risk variants for MDD, how these variants confer MDD risk remains largely unknown. Here we systematically characterize the regulatory mechanism of MDD risk variants using a functional genomics approach. By integrating chromatin immunoprecipitation sequencing (ChIP-Seq) (from human brain tissues or neuronal cells) and position weight matrix (PWM) data, we identified 34 MDD risk SNPs that disrupt the binding of 15 transcription factors (TFs)...
March 25, 2020: Molecular Psychiatry
Mami Yasukawa, Yoshinari Ando, Taro Yamashita, Yoko Matsuda, Shisako Shoji, Masaki Suimye Morioka, Hideya Kawaji, Kumiko Shiozawa, Mitsuhiro Machitani, Takaya Abe, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Yasuhide Furuta, Tadashi Kondo, Mikako Shirouzu, Yoshihide Hayashizaki, Shuichi Kaneko, Kenkichi Masutomi
The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities...
March 25, 2020: Nature Communications
Angela Lek, Yuanfan Zhang, Keryn G Woodman, Shushu Huang, Alec M DeSimone, Justin Cohen, Vincent Ho, James Conner, Lillian Mead, Andrew Kodani, Anna Pakula, Neville Sanjana, Oliver D King, Peter L Jones, Kathryn R Wagner, Monkol Lek, Louis M Kunkel
The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4 , the FSHD causal gene that encodes the highly cytotoxic DUX4 protein...
March 25, 2020: Science Translational Medicine
Victoria Jorgensen, Jingxun Chen, Helen Vander Wende, Devon Harris, Alicia McCarthy, Shane Breznak, Siu Wah Wong-Deyrup, Yuzhang Chen, Prashanth Rangan, Gloria Ann Brar, Eric M Sawyer, Leon Y Chan, Elçin Ünal
Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase ( Adh ) gene in Drosophila melanogaster Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated...
March 25, 2020: G3: Genes—Genomes—Genetics
Cheng Xiao, Miao Yu, Jieying Liu, Han Wu, Mingqun Deng, Qian Zhang, Xinhua Xiao
Familial partial lipodystrophy type 2 (FPLD2) is a rare autosomal dominant metabolic disorder caused by heterozygous mutations in the LMNA gene, which encodes for the lamin A/C. Although multiple mutations have been reported in FPLD2 patients, the mechanism remains unclear due to the lack of cellular models for the disease. We previously have generated an iPSC line (PUMCHi001-A) from a FPLD2 patient with a heterozygous R349W mutation in the LMNA gene. Here we genetically corrected the R349W mutation in the LMNA gene using CRISPR/Cas9 technology to generate an isogenic control, which was an ideal control to exclude differences in genetic background between individuals while investigating the pathogenesis of the mutation in the disease...
March 14, 2020: Stem Cell Research
Wengting Yang, Siyu Chen, Yuxin Cheng, Na Zhang, Yanxing Ma, Wei Wang, Hainan Tian, Yingying Li, Saddam Hussain, Shucai Wang
ABA regulates abiotic stress tolerance in plants via activating/repressing gene expression. However, the functions of many ABA response genes remained unknown. C/VIFs are proteinaceous inhibitors of the CWI and VI invertases. We report here the involvement of C/VIF1 in regulating ABA response and salt tolerance in Arabidopsis. We found that the expression level of C/VIF1 was increased in response to ABA treatment. By using CRISPR/Cas9 gene editing, we generated transgene-free c/vif1 mutants. We also generated C/VIF1 overexpression plants by expressing C/VIF1 under the control of the 35S promoter...
March 26, 2020: Plant Signaling & Behavior
Chuanmin Qiao, Weiwei Liu, Haoyun Jiang, Maozhang He, Qiang Yang, Yuyun Xing
p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells...
March 26, 2020: Cell Cycle
Diljith Thonnekottu, Debarati Chatterjee
The functional application of RNA-guided CRISPR associated Cas9 protein, a bacterial immune system-based protein complex via which in vivo, highly specific, and well regulated, gene editing processes are being monitored at an unprecedented level, has led to a remarkable progress in genetic engineering and technology. The complicated in vivo process of genome interrogation followed by gene editing by the Cas9 complex was recently captured by Knight et al. [ Science, 350, 825, 2015 ] by an elegant single-particle tracking method aided by the two-photon Fluorescence Correlation Spectroscopic (FCS) technique...
March 26, 2020: Journal of Physical Chemistry. B
Jianle Ren, Haibao Wang, Lei Zhou, Xinna Ge, Xin Guo, Jun Han, Hanchun Yang
The newly emerged pseudorabies virus (PRV) novel variants can escape from the immunity induced by the classical vaccine Bartha-K61. Here we investigated the underlying mechanisms by constructing chimeric mutants between epidemic strain HB1201 and the Bartha-K61 vaccine. Our analyses focused on three viral envelope glycoproteins, namely gB, gC, and gD, as they exhibit remarkable genetic variations and are also involved in induction of protective immunity. The corresponding genes were swapped reciprocally either individually or in combination by using CRISPR/Cas9 technology and homologous recombination...
2020: Frontiers in Microbiology
Veronika Kozlova, Aneta Ledererova, Adriana Ladungova, Helena Peschelova, Pavlina Janovska, Aleksander Slusarczyk, Joanna Domagala, Pavel Kopcil, Viera Vakulova, Jan Oppelt, Vitezslav Bryja, Michael Doubek, Jiri Mayer, Sarka Pospisilova, Michal Smida
Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20...
2020: PloS One
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