UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

John Gagnon

TBA
Date: 
November 29, 2017
Where: 
HSW 1057 at noon

Stephen Floor

Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein
Weijin Xu, Reazur Rahman, Michael Rosbash
bioRxiv preprint first posted online Jun. 27, 2017; doi: http://dx.doi.org/10.1101/156828. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC 4.0 International license.
November 1, 2017
Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts, 02453 USA.
We previously developed TRIBE, a method for the identification of cell-specific RNA binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA editing enzyme ADAR and performs Adenosine-to-Inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.
Date: 
November 15, 2017
Where: 
HSW 1057 at noon

Hui Li

RNA targeting with CRISPR–Cas13
Abudayyeh OO, Gootenberg J, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F.
Nature. 2017 Oct 4. doi: 10.1038/nature24049. [Epub ahead of print]
October 4, 2017
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts 02139, USA. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California 94305, USA. Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
Date: 
November 1, 2017
Where: 
HSW 1057 at noon

Theodore Roth

In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting
Chen X1, Janssen JM1, Liu J1, Maggio I1, 't Jong AEJ1, Mikkers HMM1, Gonçalves MAFV2.
Nat Commun. 2017 Sep 22;8(1):657. doi: 10.1038/s41467-017-00687-1.
September 22, 2017
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands. Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands. [email protected]
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
Date: 
November 8, 2017
Where: 
HSW 1057 at noon

Anthony Jose - ThermoFisher

PrimeFlow: Simultaneous detection of (mi)RNA and Protein by Flow Cytometry
Date: 
October 25, 2017
Where: 
HSW 1057 at noon

John Gagnon

Canceled (speaker is sick)
Date: 
October 18, 2017
Where: 
HSW 1057 at noon

Leonardo Ramos Ferreira

m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways
Li HB1, Tong J1,2, Zhu S1, Batista PJ3, Duffy EE4,5, Zhao J1,6, Bailis W1, Cao G1,2, Kroehling L1, Chen Y1,7, Wang G1, Broughton JP3, Chen YG3, Kluger Y6, Simon MD4,5, Chang HY3, Yin Z2, Flavell RA1,8.
Nature. 2017 Aug 17;548(7667):338-342. doi: 10.1038/nature23450. Epub 2017 Aug 9.
August 17, 2017
Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. The First Affiliated Hospital, Biomedical Translational Research Institute and Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Jinan University, Guangzhou 510632, China. Center for Dynamic Regulomes, Stanford University, Stanford, California 94305, USA. Department of Molecular Biophysics &Biochemistry, Yale University, New Haven, Connecticut 06511, USA. Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. Institute of Surgical Research, Daping Hospital, the Third Military Medical University, Chongqing 400038, China. Howard Hughes Medical Institute, Chevy Chase, Maryland 20815-6789, USA.
N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.
Date: 
October 11, 2017
Where: 
HSW 1057 at noon

Carol Oxford - ThermoFisher

Tips and Techniques for Optimizing Multiparameter Flow Cytometry Panels
Multiparameter flow cytometry continues to become more complex as new dyes are developed not only for surface immunophenoytping, but for functional assays, intracellular protein expression, and now gene expression. As more number of parameters are included, characterization of instrument performance and understanding of dye interactions becomes critical for detection of dimly expressed markers. Optimization of instrument voltages, and understanding of which detectors should be used to make the most sensitive measurements is critical. Instrument characterization and measurement of spillover spreading can make panel design easier, and optimize sensitivity of flow cytometry experiments.
Date: 
October 4, 2017
Where: 
HSW 1057 at noon

Mary Ann Santos

Accelerating Discovery with CRISPR Editing
Learn how genomic engineering can help build your next breakthrough! Built on 20 years of industry-leading innovation, our comprehensive genome engineering portfolio can help you meet your research needs. Together with an overview of the entire genome engineering workflow, we will focus on how to utilize the newest technologies of the CRISPR/Cas9 system to accelerate your discovery research. * The various CRISPR-Cas9 formats available today and which is best for your specific application * Transfection optimization for efficient CRISPR-Cas9 delivery * Donor synthesis for knock-in experiments * The latest easy-to-use assays for detecting and enriching cells with edited genomes * How to troubleshoot your CRISPR experiments to achieve higher efficiencies with minimal off-target effects * CRISPR lentiviral and purified gRNA libraries for screening applications * Case study of a custom cell line engineering project (knock out) Don’t miss our discussion regarding CRISPRs. For more information about this event, please contact: Jonas Ruiz [email protected] or Mary Ann Santos [email protected]
Date: 
May 31, 2017
Where: 
HSW 1057 at noon

Maryia Barnett

Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9
Niu D1,2, Wei HJ3,4, Lin L5, George H1, Wang T1, Lee IH1, Zhao HY3, Wang Y6, Kan Y1, Shrock E7, Lesha E1, Wang G1, Luo Y5, Qing Y3,4, Jiao D3,4, Zhao H3,4, Zhou X6, Wang S8, Wei H6, Güell M1, Church GM1,7,9, Yang L10.
Science. 2017 Aug 10. pii: eaan4187. doi: 10.1126/science.aan4187. [Epub ahead of print]
eGenesis, Inc., Cambridge, MA 02139, USA. College of Animal Sciences, Zhejiang University, Hangzhou 310058, China. State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China. College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China. Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark. Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, 400038, P. R. China. Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Research Institute of Shenzhen Jinxinnong Technology CO., LTD., Shenzhen 518106, China. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138, USA. eGenesis, Inc., Cambridge, MA 02139, USA. [email protected]
Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concern on pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. Earlier, we demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. Here, we confirmed that PERVs infect human cells, and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.
Date: 
September 27, 2017
Where: 
HSW 1057 at noon