UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Ryan Wagner

Engineered Cpf1 variants with altered PAM specificities
Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F.
Nat Biotechnol. 2017 Jun 5. doi: 10.1038/nbt.3900. [Epub ahead of print]
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. Department of Biological Engineering, Massachusetts Institute of Technology Cambridge, Massachusetts, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, Massachusetts, USA. Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts, USA. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan. JST, PRESTO, Tokyo, Japan. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.
Date: 
June 14, 2017
Where: 
HSW 1057 at noon

Kol Jia Yong (UPDATE: New Article)

High-resolution interrogation of functional elements in the noncoding genome
Neville E. Sanjana1,2,*,†,‡, Jason Wright1,2,†, Kaijie Zheng1,2, Ophir Shalem1,2, Pierre Fontanillas1, Julia Joung1,2, Christine Cheng1,3, Aviv Regev1,3, Feng
Science. 2016 Sep 30;353(6307):1545-1549.
September 30, 2016
1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. [email protected] [email protected] 2Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 3Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. 4Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. Howard Hughes Medical Institute, David H. Koch Institute of Integrative Cancer Biology, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ~18,000 single guide RNAs targeting >700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.
Date: 
February 22, 2017
Where: 
HSW 1057 at noon

Vanille Greiner

m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition
Zhao BS, Wang X, Beadell AV, Lu Z, Shi H, Kuuspalu A, Ho RK, He C.
Nature. 2017 Feb 23;542(7642):475-478. doi: 10.1038/nature21355. Epub 2017 Feb 13.
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, Illinois 60637, USA. Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.
Date: 
June 7, 2017
Where: 
HSW 1057 at noon

Malin Akerblom

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis
Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O, Fatica A, Santini T, Andronache A, Wade M, Laneve P, Rajewsky N, Bozzoni I.
Mol Cell. 2017 Apr 6;66(1):22-37.e9. doi: 10.1016/j.molcel.2017.02.017. Epub 2017 Mar 23.
Department of Biology and Biotechnology Charles Darwin and IBPM, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy. Center for Genomic Science of [email protected], Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, 20139 Milan, Italy. Berlin Institute for Medical Systems Biology, Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. Department of Biology and Biotechnology Charles Darwin and IBPM, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy; Center for Life Nano [email protected], Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy; Institut Pasteur Italy, Fondazione Cenci-Bolognetti, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. Electronic address: [email protected]
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.
Date: 
May 24, 2017
Where: 
HSW 1057 at noon

Kol Jia Yong

Impact of cytosine methylation on DNA binding specificities of human transcription factors
Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schübeler D, Vinson C, Taipale J.
Science. 2017 May 5;356(6337). pii: eaaj2239. doi: 10.1126/science.aaj2239.
Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 141 83 Stockholm, Sweden. Genome-Scale Biology Program, Post Office Box 63, FI-00014 University of Helsinki, Helsinki, Finland. Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Room 3128, Building 37, Bethesda, MD 20892, USA. European Synchrotron Radiation Facility, 38043 Grenoble, France. Friedrich-Miescher-Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland. Faculty of Science, University of Basel, Petersplatz 1, 4003 Basel, Switzerland. Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 141 83 Stockholm, Sweden. [email protected]
The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.
Date: 
May 17, 2017
Where: 
HSW 1057 at noon

Maryia Barnett

CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome
Tyler S Klann, Joshua B Black, Malathi Chellappan, Alexias Safi, Lingyun Song, Isaac B Hilton, Gregory E Crawford, Timothy E Reddy & Charles A Gersbach
Nat Biotechnol. 2017 Apr 3. doi: 10.1038/nbt.3853. [Epub ahead of print]
Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA. Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA. Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA. Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA. Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.
Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.
Date: 
May 10, 2017
Where: 
HSW 1057 at noon

Leonardo Ramos Ferreira

Nucleic acid detection with CRISPR-Cas13a/C2c2
Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F.
Science. 2017 Apr 28;356(6336):438-442. doi: 10.1126/science.aam9321. Epub 2017 Apr 13.
April 28, 2017
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Center for Systems Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. Department of Immunology and Infectious Disease, Harvard School of Public Health, Boston, MA 02115, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. [email protected] [email protected]
Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
Date: 
May 3, 2017
Where: 
HSW 1057 at noon

Roman Camarda

UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene
Williamson L, Saponaro M, Boeing S, East P, Mitter R, Kantidakis T, Kelly GP, Lobley A, Walker J, Spencer-Dene B, Howell M, Stewart A, Svejstrup JQ.
Cell. 2017 Feb 23;168(5):843-855.e13. doi: 10.1016/j.cell.2017.01.019. Epub 2017 Feb 16.
February 23, 2017
Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK. Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK; Institute of Cancer and Genomic Sciences, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK. Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK; Bioinformatics and Biostatistics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Bioinformatics and Biostatistics, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Experimental Histopathology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. High Throughput Screening Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Mechanisms of Transcription Laboratory, The Francis Crick Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK. Electronic address: [email protected]
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Date: 
April 26, 2017
Where: 
HSW 1057 at noon

Hui Li

Visualizing the secondary and tertiary architectural domains of lncRNA RepA
Liu F1,2, Somarowthu S1, Pyle AM1,2,3.
Nat Chem Biol. 2017 Mar;13(3):282-289. doi: 10.1038/nchembio.2272. Epub 2017 Jan 9.
March 1, 2017
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. Department of Chemistry, Yale University, New Haven, Connecticut, USA.
Long noncoding RNAs (lncRNAs) are important for gene expression, but little is known about their structures. RepA is a 1.6-kb mouse lncRNA comprising the same sequence as the 5' region of Xist, including A and F repeats. It has been proposed to facilitate the initiation and spread of X-chromosome inactivation, although its exact role is poorly understood. To gain insight into the molecular mechanism of RepA and Xist, we determined a complete phylogenetically validated secondary-structural map of RepA through SHAPE and DMS chemical probing of a homogeneously folded RNA in vitro. We combined UV-cross-linking experiments with RNA modeling methods to produce a three-dimensional model of RepA functional domains demonstrating that tertiary architecture exists within lncRNA molecules and occurs within specific functional modules. This work provides a foundation for understanding of the evolution and functional properties of RepA and Xist and offers a framework for exploring architectural features of other lncRNAs.
Date: 
April 19, 2017
Where: 
HSW 1057 at noon

Gabriel Eades

Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions
Kyuho Han, Edwin E Jeng, Gaelen T Hess, David W Morgens, Amy Li & Michael C Bassik
Nature Biotechnology
March 20, 2017
Department of Genetics, Stanford University, Stanford, California, USA. Program in Cancer Biology, Stanford University, Stanford, California, USA. Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California, USA.
Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single guide RNA (sgRNA) libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations in high-throughput and a CRISPR-based tool to dissect functional GI networks.
Date: 
April 12, 2017
Where: 
HSW 1057 at noon