DOCSLIB.ORG

WO 2017/054721 Al 6 April 2017 (06.04.2017) P O P C T

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/054721 Al 6 April 2017 (06.04.2017) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/82 (2006.01) C12N 15/113 (2010.01) kind of national protection available): AE, AG, AL, AM, A01H 5/00 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/CN2016/100533 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 28 September 2016 (28.09.201 6) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (25) Filing Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (26) Publication Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 2015 1063 1450.5 (84) Designated States (unless otherwise indicated, for every 29 September 2015 (29.09.2015) CN kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: INSTITUTE OF GENETICS AND DEVEL¬ TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, OPMENTAL BIOLOGY, CHINESE ACADEMY OF TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, SCIENCES [CN/CN]; No. 1 West Beichen Road, Chaoy- DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, ang District, Beijing 100101 (CN). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors: XIE, Qi; No. 1 West Beichen Road, Chaoyang GW, KM, ML, MR, NE, SN, TD, TG). District, Beijing 100101 (CN). YAN, Liuhua; No. 1 West Beichen Road, Chaoyang District, Beijing 100101 (CN). Published: WEI, Shaowei; No. 1 West Beichen Road, Chaoyang Dis — with international search report (Art. 21(3)) trict, Beijing 100101 (CN). YANG, Weicai; No. 1 West Beichen Road, Chaoyang District, Beijing 100101 (CN). — before the expiration of the time limit for amending the LI, Hongju; No. 1 West Beichen Road, Chaoyang District, claims and to be republished in the event of receipt of Beijing 100101 (CN). amendments (Rule 48.2(h)) (74) Agent: NTD PATENT AND TRADEMARK AGENCY — with sequence listing part of description (Rule 5.2(a)) LIMITED; 10th Floor, Block A, Investment Plaza, 27 Jin- rongdajie, Xicheng District, Beijing 100033 (CN). o o (54) Title: A CRISPR/Cas9 System for high efficient site-directed altering of plant genomes (57) Abstract: Cassettes comprising a YAO promoter operably linked to at least one nucleotide sequence encoding a nuclease, vec - tors comprising the same are provided. A system for altering a plant genome comprising a nucleotide sequence encoding a nuclease operably linked to a YAO promoter and a method to alter the target nucleic acid molecule by using the system are provided. Plants, progeny and seeds thereof having such altered target nucleic acid molecules are also provided. A CRISPR/Cfl«9 System for high efficient site-directed altering of plant genomes REFERENCE TO RELATED APPLICATIONS This application claims priority to previously filed and co-pending application CN1 05177038, filed September 29, 201 5, the contents of which are incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on September 26, 2016, is named P12040WO00_SL.txt and is 105,189 bytes in size. TECHNICAL FIELD The present invention relates to the field of biotechnology, particularly a CRISPR/ Cas9 system for high efficient site-directed altering of plant genomes. BACKGROUND The realization of high efficient, site-directed altering for plant genomes is of great significance to study the functions of plant genes. At present, gene modification techniques, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and CRISPR C etc., have been widely used in scientific research, wherein the CRISPR C x technique is a recently developed gene modification technique. The CRISPR/C system is an acquired immune system presently discovered which exists in most bacteria and all archaea to eliminate extraneous plastids or phages, and to leave extraneous gene fragments in autologous genomes as "memories". Different forms of deletions or insertions have been created at target fragments by editing organism genomes with a CRISPR/ Cas9 system, which has been successfully used in organisms such as Homo sapiens cell lines, Danio rerio, Rattus norvegicus, Mus musculus, Drosophila melanogaster etc. In the field of plants, this technique has also been used in plants such as Arabidopsis thaliana, Oryza saliva L., Zea maysL., Nicotiana tabacum, Lycopersicon esculentum etc., but the editing efficiency of the existing CRISPR C x system is low. At present, the promoters used for driving nucleases in these systems, such as the Cas9 gene expressionor Fokl gene expression are mostly are CMV 35S promoter and Ubiquitin promoter, but previous studies have demonstrated that, the editing efficiencies of Cas9 to plant genomes driven by the both are low. It can be seen that, for improving the editing efficiencies, it is especially important to select suitable promoters for driving the expression of Cas9 gene. SUMMARY OF THE INVENTION Increased frequency of gene altering is provided by use of a YAO promoter. When used with a gene editing system such as CRISPR C , TALEN or Zinc finger nucleases, the frequency of gene editing is increased compared to use of a promoter that is not the YAO promoter and in particular compared to using the 35S promoter. In one embodiment the YAO promoter is operably linked with a nucleic acid molecule that encodes a Cas9 or Fokl polypeptide. Gene editing frequency is increased to at least 75% or more and up to 90%, 95% or more. The frequency of gene editing of a targeted nucleic acid molecule is at least five times, 18 times or higher than when using a 35S promoter. The increased gene frequency is also provided in progeny of a plant into which a cassette is introduced comprising the YAO promoter driving a nuclease such as the Cas9 or Fokl nucleic acid molecule. Cassettes, vector, edited plants and cells are also provided. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A and IB is a diagram showing structure of the CRISPR C z binary vectors for Arabidopsis transformation. The hSpCas9 cassette is driven by the 35S (see Figure 1A) or YAO (Figure IB) promoter, while sgRNA is controlled by the AtU6-26 promoter. NLS refers to the nuclear localization sequence. Figure 2 is a gel showing RFLP detect the site-directed editing effects of 35S:Cas91 6-26- sgRNA system and pYAO:Cas9lAtU6-26-sgRNA system on endogenous gene BRI1 of Arabidopsis thaliana. Here, M is a DNA Marker; Lanes 1-23 in Figure 2A are electrophoresis results of PCR products of Tl generation Arabidopsis thaliana introduced with 35S:Cas9iAt 6- 26-sgRNA system after EcoR V enzyme cleavage, Lanes 1-21 in Figure 2B are electrophoresis results of PCR products of Tl generation Arabidopsis thaliana introduced with system after EcoR V enzyme cleavage; and Col-0 is electrophoresis result of PCR products of wild type Arabidopsis thaliana after EcoR V enzyme cleavage. Figure 3A-C are graphs showing sequencing analysis for site-directed editing effects of 35S:Cas9/AtXJ6-26-sgRNA system and ? :C /AtU6-26-sgRNA system on endogenous gene BRI1 of T l generation Arabidopsis thaliana. Here, Figure 3A is a peak profile of sequencing for PCR products of 35S:hSpCas9-BRIl-sgKNA system vs. 35S-6-TI ; Figure 3B is a peak profile of sequencing for PCR products ofpYAO:hSpCas9-BRIl -sgRNA system vs. pYAO- 16-T1 ; Figure 3C is a peak profile of sequencing for PCR products of pYAO:hSpCas9-BRIl - sgRNA system vs. ρΥΑΟ- -Ί \ Figure 4A Figure 4A shows editing forms of 35S-6-TI and ρΥΑΟ-\6- Ύ\ at target sites of BRI1 gene (SEQ ID NOS 75-77, respectively, in order of appearance); and Figure 4B shows editing forms ΐ ρΥΑΟ- -Ί \ at target sites of BRI1 gene (SEQ ID NOS 75, 78, 79, 77 and 80, respectively, in order of appearance); WT represents the nucleotide sequences of wild-type Arabidopsis thaliana at the target sites, "D" represents the sequences subjected to deletion mutations, "+" represents the sequences subjected to insertion mutations, and the numbers behind "D/+" represent the amount of deleted or inserted nucleotides. Figure 5 shows representative sequences of several mutant alleles of BRI1 identified from the p :hSpC s -BRIl -sgRNA Tl transgenic plant line 4 and line 2 1 (SEQ ID NOS 81-86, 83 and 87, respectively, in order of appearance). The wild-type sequence is shown at the top with the PAM sequence in bold. Figure 6A is a gel showing RFLP analysis of genomic DNA from the pYAO.hSpCas9 -PDS3- sgRNA Tl plants. Figure 6B shows representative sequences of several mutant alleles of PDS3 identified from a pYAO:hSpCas9 - O S -sgRNA Tl transgenic plant (SEQ ID NOS 88-96, 91, 94, 92, 97, 9 1 and 98, respectively, in order of appearance).
Recommended publications
  • The Role of NF-Κb and C/EBP Factors During Pathogen-Mediated

    The Role of NF-Κb and C/EBP Factors During Pathogen-Mediated

    The role of NF-B and C/EBP factors during pathogen-mediated activation of bovine interleukin 8 and beta-defensin in mammary epithelial cells Inaugural dissertation for the academic degree Doctor rerum naturalium of Mathematisch-Naturwissenschaftlichen Fakultät Universität Rostock By Shuzhen Liu (M. Sc.), born on March-02-1972, in Shanxi Province, China From the Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere in Dummerstorf Rostock 2009 URN: urn:nbn:de:gbv:28-diss2009-0185-5 Dean: Prof. Dr. Hendrik Schubert Reviewers: 1. Prof. Dr. Hans-Martin Seyfert Research Unit molecular biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany 2. Prof. Dr. Dieter G. Weiss Division of animal Physiology, Institute of Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Strasse 3, 18059 Rostock, Germany 3. PD Dr. Ulrike Gimsa Research Unit Behavioural Physiology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Date of defense: October 19th, 2009 Table of Contents TABLE OF CONTENTS 1. INTRODUCTION..................................................................................................................1 1.1 Mastitis as a challenge in general immunology..................................................................1 1.2 Innate immunity of the bovine mammary gland ................................................................2 1.3 Toll-like receptors (TLRs): main receptors perceiving the pathogen
  • CLONING, EXPRESSION ANALYSIS, and TRANSFORMATION VECTOR CONSTRUCTION of DAM HOMOLOGS in PEACH and POPLAR Yuhui Xie Clemson University, Yxie@Clemson.Edu

    CLONING, EXPRESSION ANALYSIS, and TRANSFORMATION VECTOR CONSTRUCTION of DAM HOMOLOGS in PEACH and POPLAR Yuhui Xie Clemson University, [email protected]

    Clemson University TigerPrints All Theses Theses 1-2011 CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR Yuhui Xie Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Part of the Plant Biology Commons Recommended Citation Xie, Yuhui, "CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR" (2011). All Theses. 1146. https://tigerprints.clemson.edu/all_theses/1146 This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. CLONING, EXPRESSION ANALYSIS, AND TRANSFORMATION VECTOR CONSTRUCTION OF DAM HOMOLOGS IN PEACH AND POPLAR _______________________________________________ A Thesis Presented to the Graduate School of Clemson University ________________________________________________ In Partial Fulfillment of the Requirement for the Degree Master of Science Plant and Environmental Sciences _________________________________________________ by Yuhui Xie August 2011 ________________________________________________ Accepted by: Dr. Douglas G. Bielenberg, Committee Chair Dr. Haiying Liang Dr. Hong Luo ABSTRACT Genetic fine mapping and sequencing of the EVG locus in peach [Prunus persica (L.) Batsch] identified six tandem arrayed Dormancy-Associated MADS-box (DAM) genes as candidates for regulating growth cessation and terminal bud formation in the non-dormant evergrowing (evg) mutant. Since the mutant is lacking expression of six genes in the mapped locus, further functional analysis is needed to narrow the list of gene candidates for the non-dormant evg phenotype. Here I report three sets of experiments designed to functionally test DAM genes in peach and their homologs in a model tree, hybrid poplar.
  • Enzymes in Cloning Part I

    Enzymes in Cloning Part I

    ˹̀/˺̀/˺̊˼̊ ENZYMES IN CLONING PART I Dr.Sarookhani ˺ ˹̀/˺̀/˺̊˼̊ Cloning --aa definition •• From the Greek --klon,klon, a twig •• An aggregate of the asexually produced progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody) •• An individual grown from a single somatic cell of its parent & genetically identical to it •• Clone: a collection of molecules or cells, all identical to an original molecule or cell Dr.Sarookhani ˻ ˹̀/˺̀/˺̊˼̊ Different types of Cloning 1. Reproductive Cloning 2. Therapeutic Cloning 3. Recombinant DNA Technology or DNA Cloning Dr.Sarookhani ˼ ˹̀/˺̀/˺̊˼̊ DNA CLONING A method for identifying and purifying a particular DNA fragment (clone) of interest from a complex mixture of DNA fragments, and then producing large numbers of the fragment (clone) of interest. Dr.Sarookhani ̊ ˹̀/˺̀/˺̊˼̊ What is genetic engineering • Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. • Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all. Dr.Sarookhani ̋ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̌ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̀ ˹̀/˺̀/˺̊˼̊ Genomic Library Dr.Sarookhani ́ ˹̀/˺̀/˺̊˼̊ Dr.Sarookhani ̂ ˹̀/˺̀/˺̊˼̊ Basic steps in genetic engineering 1.
  • Cyclic Digestion and Ligation-Mediated PCR Used For

    Cyclic Digestion and Ligation-Mediated PCR Used For

    www.nature.com/scientificreports OPEN Cyclic Digestion and Ligation- Mediated PCR Used for Flanking Sequence Walking Dong Yu1,2,5, Tianshun Zhou2,4,5, Xuewu Sun2,3, Zhizhong Sun2, Xiabing Sheng1,2, Yanning Tan2, Ling Liu2,4, Ning Ouyang2,4, Ke Xu2, Kaibing Shi2, Guilong Yuan2, Jia Ding2, Meijuan Duan3* & Dingyang Yuan1,2,3,4* Ligation-mediated PCR (LM-PCR) is a classical method for isolating fanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and fexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 diferent adapters at low cost. Using this method, the fanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efcient and fexible method for identifying the fanking sequences in transgenic rice and Arabidopsis thaliana. Identifcation of fanking sequences has ofen been employed to determine the location of T-DNA insertion in genomic DNA. Methods to obtain fanking sequencea include inverse PCR1, randomly primed PCR2–5, and ligation-mediated PCR6–8. Inverse PCR, the earliest fanking cloning technique, has a low efciency and is limited by the rate of self-ligation and amplifcation range of DNA polymerases9.
  • CRISPR Foki Dead Cas9 System: Principles and Applications in Genome Engineering

    CRISPR Foki Dead Cas9 System: Principles and Applications in Genome Engineering

    cells Review CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering Maryam Saifaldeen y, Dana E. Al-Ansari y , Dindial Ramotar * and Mustapha Aouida * College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha P.O.Box 34110, Qatar; [email protected] (M.S.); [email protected] (D.E.A.-A.) * Correspondence: [email protected] (D.R.); [email protected] (M.A.) These authors contributed equally to this work. y Received: 27 September 2020; Accepted: 19 November 2020; Published: 21 November 2020 Abstract: The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms.
  • The Application of Transcription Activator-Like Effector Nucleases for Genome Editing in C

    The Application of Transcription Activator-Like Effector Nucleases for Genome Editing in C

    Methods 68 (2014) 389–396 Contents lists available at ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth The application of transcription activator-like effector nucleases for genome editing in C. elegans ⇑ ⇑ Peishan Yi a,b, Wei Li c, , Guangshuo Ou d, a National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China b University of Chinese Academy of Sciences, Beijing 100049, China c School of Medicine, Tsinghua University, Beijing 100084, China d Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China article info abstract Article history: The nematode Caenorhabditis elegans has been a powerful model system for biomedical research in the Received 31 December 2013 past decades, however, the efficient genetic tools are still demanding for gene knockout, knock-in or con- Revised 14 April 2014 ditional gene mutations. Transcription activator-like effector nucleases (TALENs) that comprise a Accepted 17 April 2014 sequence-specific DNA-binding domain fused to a FokI nuclease domain facilitate the targeted genome Available online 26 April 2014 editing in various cell types or organisms. Here we summarize the recent progresses and protocols using TALENs in C. elegans that generate gene mutations and knock-ins in the germ line and the conditional Keywords: gene knockout in somatic tissues. Caenorhabditis elegans Ó 2014 Elsevier Inc. All rights reserved. Transcription activator-like effector nucleases (TALENs) Mutation Conditional gene knockout 1. Introduction length at the site of the break, whereas HDR can generate precisely specified changes at the target site when a homologous DNA The newly-developed transcription activator-like effector template is provided.
  • James Alan Tunaley

    James Alan Tunaley

    Structure and Activity Investigations of the Cell Fate Determinant, SpoIIE, from Bacillus subtilis. James Alan Tunaley Thesis Submitted for the Degree of Doctor of Philosophy University of York Department of Chemistry September 2013 Abstract For many years the Gram positive bacterium Bacillus subtilis has been a model organism for prokaryotic cell and molecular biology. The asymmetric cell division which B. subtilis undergoes during sporulation is a simple system by which to study the process of cell differentiation. Sporulation is governed by a series of genetic temporal and spatial controls. Gene regulation brought about by a series of σ factors and transcriptional regulators is coupled to key morphological stages or checkpoints. σF initiates the first step in a cascade of complex genetic control which eventually produces a resilient endospore. The activation of σF, the first compartment-specific sigma factor, in the forespore and its regulation through interaction between three proteins; SpoIIAA, SpoIIAB and SpoIIE, is of particular interest. SpoIIE, a protein phosphatase which binds to the asymmetric division septum, is a crucial factor in the selective activation of σF in the forespore. Of three putative domains in SpoIIE only the C-terminal PP2C phosphatase domain has been structurally characterised. The central domain, domain II, of SpoIIE has been assigned a role in interaction with the cell division machinery; however mutational studies have shown that, in addition, this domain is also responsible for the regulation of phosphatase activity. This work describes the isolation and characterisation of three new fragments of SpoIIE containing elements of the central cytoplasmic domain of SpoIIE. These include a fragment found to accurately represent the N-terminal solubility limit of domain II which shows a high degree of oligomeric character.
  • Review Type II Restriction Endonucleases

    Review Type II Restriction Endonucleases

    CMLS, Cell. Mol. Life Sci. 62 (2005) 685–707 1420-682X/05/060685-23 DOI 10.1007/s00018-004-4513-1 CMLS Cellular and Molecular Life Sciences © Birkhäuser Verlag, Basel, 2005 Review Type II restriction endonucleases: structure and mechanism A. Pingouda,*, M. Fuxreiterb, V.Pingouda and W.Wendea a Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, 35392 Giessen (Germany), Fax: +49 641 9935409, e-mail: [email protected] b Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, Karolina ut 29, 1113 Budapest (Hungary) Received 15 November 2004; accepted 9 December 2004 Abstract. Type II restriction endonucleases are compo- few restriction endonucleases discovered thus far do not nents of restriction modification systems that protect belong to the PD…D/ExK family of enzymes, but rather bacteria and archaea against invading foreign DNA. Most have active sites typical of other endonuclease families. are homodimeric or tetrameric enzymes that cleave DNA The present review deals with new developments in the at defined sites of 4–8 bp in length and require Mg2+ ions field of Type II restriction endonucleases. One of the for catalysis. They differ in the details of the recognition more interesting aspects is the increasing awareness of process and the mode of cleavage, indicators that these the diversity of Type II restriction enzymes. Nevertheless, enzymes are more diverse than originally thought. Still, structural studies summarized herein deal with the more most of them have a similar structural core and seem to common subtypes. A major emphasis of this review will share a common mechanism of DNA cleavage, suggest- be on target site location and the mechanism of catalysis, ing that they evolved from a common ancestor.
  • Dissertation M.Sc Lila Oubraham

    Dissertation M.Sc Lila Oubraham

    Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by M.Sc Lila Oubraham born in: Algiers, Algeria Oral-examination: 11.05.2016 Nuclease-mediated gene manipulation of factors implicated in zebrafish neurogenesis Referees: Prof. Dr. Uwe Strähle Prof. Dr. Nicholas S. Foulkes Summary Complex and differential gene expression programs give rise to several cell types that constitute the different parts of the organism. This cell fate determination is controlled by a group of proteins, named transcription regulators (TRs). Our group investigates the molecular mechanisms underlying neurogenesis using the zebrafish as a model system. To that purpose, a genome-wide analysis of TR gene expression was performed in our laboratory, and hundreds of these regulators were identified. On the basis of these initial studies, a number of TRs were selected for further characterization. For this project, two model systems for zebrafish neurogenesis were chosen: The embryonic spinal cord and the adult telencephalon. The spinal cord is considered as relatively simple and is used to understand the neural differentiation and function in vertebrates during development. Based on morpholinos knockdown experiments, two closely related genes sox1a and sox1b encoding transcription factors, were shown to play a role in the specification of a newly observed sub-type of interneurons, named V2c, in the ventral spinal cord of the zebrafish. Nevertheless, the epistatic relationship between these genes has still to be investigated. On the other hand, in the adult brain, new neurons are continuously generated from neural progenitor cells.
  • Type II Restriction Endonucleases : a Historical Perspective and More

    Type II Restriction Endonucleases : a Historical Perspective and More

    Published online 30 May 2014 Nucleic Acids Research, 2014, Vol. 42, No. 12 7489–7527 doi: 10.1093/nar/gku447 SURVEY AND SUMMARY Type II restriction endonucleases––a historical perspective and more Alfred Pingoud1,*,†, Geoffrey G. Wilson2,† and Wolfgang Wende1 1Institute of Biochemistry, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany and 2New England Biolabs Inc., 240 County Road, Ipswich, MA 01938-2723, USA Downloaded from Received January 7, 2014; Revised May 02, 2014; Accepted May 7, 2014 ABSTRACT combat viral infections, these enzymes allow unmanageable http://nar.oxfordjournals.org/ tangles of macromolecular DNA to be transformed with This article continues the series of Surveys and Sum- unsurpassable accuracy into convenient, gene-sized pieces, maries on restriction endonucleases (REases) begun a necessary first step for characterizing genomes, sequenc- this year in Nucleic Acids Research.Herewedis- ing genes, and assembling DNA into novel genetic arrange- cuss ‘Type II’ REases, the kind used for DNA anal- ments. It seems unlikely that today’s Biomedical Sciences ysis and cloning. We focus on their biochemistry: and the Biotechnology industry would have developed with- what they are, what they do, and how they do it. Type out Type II restriction enzymes, and certainly not at the II REases are produced by prokaryotes to combat startling pace we have witnessed since their discovery only at Bibliothekssystem der Universitaet Giessen on February 10, 2015 bacteriophages. With extreme accuracy, each recog- a few decades ago. nizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The INTRODUCTION discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since im- Several reviews of restriction endonucleases (REases) have pacted every corner of the life sciences.
  • Assignment 1 Part I

    Assignment 1 Part I

    1 Assignment 1 Note: Part I of the assignment is to be submitted through Blackboard (See instructions on Blackboard). Parts II, III and IV are to be submitted as a hard copy during your lab session. Part I Dilutions and Concentrations When doing your calculations, do not round off your intermediate numbers. Only round off the final answer. Your answers must be submitted to two significant figures after the decimal. For example 2.00, 0.020, or 0.0020. It is strongly recommended that you submit your answers using the web browser Firefox. You will be allowed two submissions! Use the following information to answer questions 1-6 You prepare a solution by adding the following ingredients in the order indicated: 600 mL H2O, 125 mL 1.6 M LiCl, 50 mL 20 % (m/v) MgCl2, and 25 mL 10g/L NaCl. The properties of each ingredient are as follows: LiCl: MW 200g/mole, density 1.2g/mL, density of 1.6 M soln. 1.05g/mL MgCl2: MW 150g/mole, density 1.3g/mL, density of 20% soln. 1.1g/mL NaCl: MW 35g/mole, density 1.15g/mL, density of 10g/L soln. 1.03g/mL Final solution: density: 1.25g/mL 1. What is the final molarity of MgCl2 in the solution? (0.083M) 2. What is the volume in milliliters of one part? (25 mL) 3. What is the percentage (m/m) of NaCl in the final solution? (0.025%) 4. What is the percentage (m/v) of LiCl in the final solution? (5%) 5. What is the number of parts of solvent in the final solution? (24 parts) 6.
  • Talens As a Talented and Versatile Genome Editing Tool

    Talens As a Talented and Versatile Genome Editing Tool

    ENGINEERING OF TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) FOR TARGETED GENOME EDITING BY NING SUN DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2013 Urbana, Illinois Doctoral Committee: Professor Huimin Zhao, Chair Professor Susan A. Martinis Associate Professor Fei Wang Professor Yi Lu Professor James H. Morrissey ABSTRACT In the post-genome era, one of the most important topics of research is to edit or program genomic sequences and to generate desired phenotypes. Although virus-based strategies have long been developed to for efficient gene insertion, the random or semi-random integration can disrupt certain endogenous genes and cause unpredictable phenotypes. In contrast, targeted genome editing enables researchers to tailor genomic loci in a specific manner. Applications include studying gene functions, engineering microbes for industrial fermentation, improving traits in crop plants and livestock, treating human diseases, etc. This thesis describes my efforts on engineering transcription activator-like effector (TALE) nucleases (TALENs) as an efficient tool for targeted genome editing. Targeted genome engineering relies on the introduction of a site-specific double- strand break (DSB) in a pre-determined genomic locus by a rare-cutting DNA endonuclease. Subsequent repair of this DSB by non-homologous end joining or homologous recombination generates the desired genetic modifications such as gene disruption, gene insertion, gene correction, etc. For this purpose, I have constructed TALEN architecture by fusing the DNA binding domain of TALE and a FokI non- specific DNA cleavage domain. TALEs are isolated from the plant pathogenic bacteria from the genus Xanthomonas and their DNA binding domains are composed of a series of tandem repeats.