RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins Thesis by Hatoon Baazim In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia May 2014 2 EXAMINATION COMMITTEE APPROVALS FORM The thesis of Hatoon Baazim is approved by the examination committee. Committee Chairperson: Magdy Mahfouz Committee Member: Christoph Gehring Committee Member: Samir Hamdan 3 © Approval Date May 2014 Hatoon Baazim All Rights Reserved 4 ABSTRACT RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins Hatoon Baazim Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications. 5 TABLE of CONTENTS Page Examination Committee Approvals Form ................................................................ 2 Copyright Page ......................................................................................................... 3 Abstract ..................................................................................................................... 4 Table Of Contents ..................................................................................................... 5 List Of Abbreviations ................................................................................................ 7 List Of Illustrations ................................................................................................... 8 List Of Tables ............................................................................................................ 9 1. Introduction …………………………………………………………………...... 10 1.1 Zinc Finger-Based Transcriptional Regulators ……………………….. .......... 13 1.2 TAL-based transcriptional regulators …………………….. ............................... 17 1.3 CRISPR/Cas9 Systems ……………………….. ................................................. 20 1.3.1 RNA-Guided dCas9 Transcriptional Regulation……………………….. ....... 23 1.3.2 Transcriptional Regulation In Bacteria……………………….. ...................... 24 1.3.3 Transcriptional Regulation In Eukaryotes……………………….. .................. 24 1.3.4 Development of synthetic transcriptional regulators in plants via dCas9 CRISPR Systems ………………………………………………………………… . 26 2. Materials and Methods……………………….. .................................................... 28 2.1 Plasmid Construction……………………….. .................................................... 28 2.2 Agroinfiltration……………………….. .............................................................. 29 2.3 GUS Qualitative and Quantitative assays……………………….. ..................... 30 2.4 RNA Isolation and Semi-quantitative PCR……………………….. ................... 30 6 3. Results ……………………….. ............................................................................ 32 3.1 Design and construction of dCas9-based chimeric transcriptional regulators... 32 3.2 The dCas9.TAs mediate strong transcriptional activation of a target gene ….. 35 3.3 Positional of gRNAs binding in the promoter influence transcriptional activation ……………………….. ............................................................................ 38 3.4 Transcriptional activation is enhanced by multiple gRNA…………………….. 40 3.5 The dCas9-SRDX mediates transcriptional repression of a PDS target gene …. 43 3.6 The dCas9-EDLL and dCas9-TAD mediate transcriptional activation of a PDS target gene…………………………………………………………………….......... 45 3.7 Synthetic transcriptional repressor interferes and competes with transcriptional activation machinery.……………………….. .......................................................... 47 4. Discussion……………………….. ....................................................................... 49 References……………………….. ........................................................................... 54 Supplementary information ……………………….. ................................................ 61 7 LIST OF ABBREVIATIONS GOI Gene of interest Cas9 CRISPR-associated endonuclease 9 CHS Chalcone synthase gene CRISPR Cluster Regulatory Intersperced Palindramic Repeat dCas9 dead-Cas9 (catalytically inactive Cas9) DSB double-stranded break HD Histidine, Aspartate HDR Homology directed repair KRAB Krüpple-associated box NHEJ Non-homologous end joining NI Asparagine, Isoleucine NK Asparagine, Lysine NG Asparagine, Glycine NN Asparagine, Asparagine NS Asparagine, Serine PAM Protospacer adjacent motif RT-PCR Reverse Transcription-Polymerase Chain Reaction RVD Repeat Variable Di-residue TALE Transcription Activator-like Effector ZFN Zinc Finger Nuclease 8 LIST OF ILLUSTRATIONS Page Figure 1: (RNAi) RNA interference pathway.............................................................. 12 Figure 2: Zinc-Finger DNA-binding domain ……………………………….............. 14 Figure 3: TAL effector components and DNA-binding ……………………………. 18 Figure 4: CRISPR/Cas bacterial immune defense mechanism ……………………... 21 Figure 5: A schematic representation of the dCas9 protein ……………………........ 23 Figure 6: Overview of the experimental design ……………………………………. 27 Figure 7: Schematic representation of dCas9 chimeric effector proteins …............... 33 Figure 8: Schematic representation of episomal and endogenous gRNA target sequences used in this study ………………………………………………………… 35 Figure 9: Transcriptional activation mediated by chimeric dCas9.TFs ……………. 37 Figure 10: Positional effect of dCas9 promoter binding elements on transcriptional activation of Bs3::uidA episomal target gene ………………………………………. 39 Figure 11: Synergistic effects of dCas9.TFs complexes on the overall transcriptional regulation of the Bs3 promoter …………………………………………………….... 42 Figure 12: dCas9.SRDX mediated repression of PDS endogenous target in Nicotiana benthamiana transient leaf assays …………………………………………………… 44 Figure 13: Transcriptional activation of endogenous PDS target using dCas9.TAs… 46 Figure 14: Interference analysis of the dCas9:SRDX repressor with dCas9:EDLL and dCas9:TAD transcriptional activators ………………………………………………. 48 9 LIST OF TABLES Page Table 1: RVD: Nucleotide specificities .................................................................. 18 10 1 Introduction: The ability to perturb the transcriptional patterns and interrogate gene functions in a site- specific manner will open new bioengineering possibilities with applications in basic biology and biotechnology. Since the advent of plant biotechnology, researchers have attempted to up-regulate or down-regulate the expression levels of a specific gene of interest (GOI) for functional studies. Over-expression is one of the first techniques adopted in the investigation of gene function, enhancing the expression of the GOI and then examining the resulting phenotypic effects. The first applications of over-expression in plants used cell cultures [1] and later on Arabidopsis thaliana. These experiments included the activation-tagging system [2], followed by a study on the FOX-hunting system, which involved cDNA expression under strong promoter such as the cauliflower mosaic virus 35S promoter [3]. An earlier study during 1990 aimed to over-express Chalcone synthase in Petunia flowers, using a construct containing the petunia CHS gene expressed under the control of 35S promoter [4], unexpectedly resulted in the complete and/or partial blockage of the corresponding endogenous gene expression, producing a phenotype of white and/or patterned flowers. This study introduced a new technique, henceforth termed co- suppression, which was then widely used in Arabidopsis, Drosophila and C. elegance [5,6]. Studies investigating the co-suppression mechanism of action suggested two hypotheses, the first of which suggests that the silencing occurs transcriptionally (TGS), as a result of direct interaction between the chimeric gene and its endogenous homologue, 11 which epigenetically silences the gene expression, either by methylation or chromatin modification. The second and more plausible hypothesis, suggested post-transcriptional gene silencing (PTGS), which