UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Maryia Barnett

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice
Xu C1, Qi X1, Du X1, Zou H1, Gao F1, Feng T1, Lu H1, Li S2,3, An X1, Zhang L1, Wu Y4, Liu Y2,3,5, Li N1, Capecchi MR6, Wu S7.
Proc Natl Acad Sci U S A. 2017 Jan 6. pii: 201615735. doi: 10.1073/pnas.1615735114. [Epub ahead of print]
January 6, 2017
1State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China. 2Department of Neurosurgery, Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030. 3Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030. 4Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112. 5The Senator Lloyd & B. A. Bentsen Center for Stroke Research, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX 77030. 6Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112; [email protected] [email protected] 7State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; [email protected] [email protected]
CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.
Date: 
January 25, 2017
Where: 
HSW 1057 at noon

TBD

TBA
Date: 
January 18, 2016
Where: 
HSW 1057 at noon

Gabriel Eades

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library
Shiyou Zhu, Wei Li, Jingze Liu, Chen-Hao Chen, Qi Liao, Ping Xu, Han Xu, Tengfei Xiao, Zhongzheng Cao, Jingyu Peng, Pengfei Yuan, Myles Brown, Xiaole Shirley Liu & Wensheng Wei
Nat Biotechnol. 2016 Dec;34(12):1279-1286. doi: 10.1038/nbt.3715. Epub 2016 Oct 31.
October 31, 2016
1Biodynamic Optical Imaging Center (BIOPIC), Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China. 2Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program (PTN), Peking University, China. 3Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA. 4Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 5Department of Prevention Medicine, School of Medicine, Ningbo University, Ningbo, Zhejiang, China. 6Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, Massachusetts, USA. 7Division of Molecular and Cellular Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 8Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. 9Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.
Date: 
January 11, 2017
Where: 
HSW 1057 at noon

Not in Session

Winter Break
Date: 
January 4, 2017
Where: 
HSW 1057 at noon

Canceled

N/A
Date: 
July 13, 2016
Where: 
HSW 1057 at noon

Canceled

N/A
Where: 
HSW 1057 at noon

Not in Session

Winter Break
Date: 
December 28, 2016
Where: 
HSW 1057 at noon

Not in Session

Winter Break
Date: 
December 21, 2016
Where: 
HSW 1057 at noon

Roman Camarda

LKB1 loss links serine metabolism to DNA methylation and tumorigenesis
Kottakis F1,2,3, Nicolay BN1,3, Roumane A1,2,3, Karnik R4,5,6, Gu H4,5,6, Nagle JM1,2,3, Boukhali M1,3, Hayward MC7, Li YY8,9, Chen T8,9,10, Liesa M11,12, Hammerman PS8,9,13, Wong KK8,9,10, Hayes DN7, Shirihai OS11,12, Dyson NJ1,3, Haas W1,3, Meissner A4,5,6, Bardeesy N1,2,3.
Nature. 2016 Oct 31;539(7629):390-395. doi: 10.1038/nature20132. [Epub ahead of print]
October 31, 2016
1Cancer Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA. 2Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA. 3Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA. 4Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 5Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA. 6Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA. 7UNC, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA. 8Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA. 9Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA. 10Belfer Institute for Applied Cancer Science, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA. 11Evans Center for Interdisciplinary Research, Department of Medicine, Mitochondria ARC, Boston University School of Medicine, Boston, Massachusetts 02118, USA. 12Department of Medicine, Division of Endocrinology, Diabetes and Hypertension, UCLA David Geffen School of Medicine, Los Angeles, California 90095, USA. 13Cancer Program, Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA.
Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.
Date: 
December 14, 2016
Where: 
HSW 1057 at noon

Theodore Roth

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration
Suzuki K1, Tsunekawa Y2, Hernandez-Benitez R1,3, Wu J1,4, Zhu J5,6, Kim EJ7, Hatanaka F1, Yamamoto M1, Araoka T1,4, Li Z8, Kurita M1, Hishida T1, Li M1, Aizawa E1, Guo S8, Chen S8, Goebl A1, Soligalla RD1, Qu J9,10, Jiang T6,11, Fu X5,6, Jafari M6, Esteban CR1, Berggren WT12, Lajara J4, Nuñez-Delicado E4, Guillen P4,13, Campistol JM14, Matsuzaki F2, Liu GH10,15,16,17, Magistretti P3, Zhang K8, Callaway EM7, Zhang K5,6,18,19, Belmonte JC1.
Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub 2016 Nov 16.
December 1, 2016
1Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA. 2Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan. 34700 King Abdullah University of Science and Technology (KAUST) Thuwal 23955-6900, Saudi Arabia. 4Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain. 5Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China. 6Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA. 7Systems Neurobiology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA. 8Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA. 9State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. 10University of Chinese Academy of Sciences, Beijing 100049, China. 11Guangzhou EliteHealth Biological Pharmaceutical Technology Company Ltd, Guangzhou 510005, China. 12Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA. 13Fundación Dr. Pedro Guillen, Investigación Biomedica de Clinica CEMTRO, Avenida Ventisquero de la Condesa, 42, 28035 Madrid, Spain. 14Hospital Clinic, University of Barcelona, IDIBAPS, 08036 Barcelona, Spain. 15National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 16Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou 510632, China. 17Beijing Institute for Brain Disorders, Beijing 100069, China. 18Molecular Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, China. 19Veterans Administration Healthcare System, San Diego, California 92093, USA.
Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.
Date: 
December 7, 2016
Where: 
HSW 1057 at noon