Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2020 CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli Nicholas John Backes Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Recommended Citation Backes, Nicholas John, "CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli" (2020). Graduate Theses and Dissertations. 18089. https://lib.dr.iastate.edu/etd/18089 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli by Nicholas Backes A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Microbiology Program of Study Committee: Gregory Phillips, Major Professor Jinoh Kim Thomas Mansell Dipali Sashital Stephan Schmitz-Esser The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2020 Copyright © Nicholas Backes, 2020. All rights reserved. ii TABLE OF CONTENTS Page LIST OF FIGURES ....................................................................................................................... vi LIST OF TABLES ....................................................................................................................... viii ACKNOWLEDGMENTS ............................................................................................................. ix ABSTRACT ................................................................................................................................... xi CHAPTER 1. GENERAL INTRODUCTION ................................................................................1 Motivation ................................................................................................................................. 1 Thesis Objectives ....................................................................................................................... 4 Thesis Organization ................................................................................................................... 4 CHAPTER 2. REPURPOSING CRISPR-CAS SYSTEMS AS GENETIC TOOLS IN ENTEROBACTERIACEAE ...........................................................................................................6 Abstract ...................................................................................................................................... 6 Introduction ............................................................................................................................... 6 The Basics of CRISPR-Cas ....................................................................................................... 9 CRISPR-Cas Tools for Bacteria .............................................................................................. 13 CRISPR-Cas Genome Editing ................................................................................................. 14 Alternative Bacterial Genome Editing Strategies Using CRISPR-Cas .............................. 22 CRISPR Modulation of Gene Expression ............................................................................... 25 Programmable Gene Repression by CRISPRi ................................................................... 25 Synthetic CRISPR Arrays for Multi-gene Targeting ......................................................... 27 Bacterial Cell Processes ..................................................................................................... 29 Bacterial Pathogens ............................................................................................................ 30 Synthetic Biology ............................................................................................................... 31 Metabolic Engineering ....................................................................................................... 32 Programmable Gene Activation by CRISPRa.................................................................... 32 Genetic Screens .................................................................................................................. 35 Expanding CRISPR-Cas Systems ........................................................................................... 37 Designing CRISPRi Experiments ............................................................................................ 38 Guide RNA Design ............................................................................................................ 39 Guide RNA Expression ...................................................................................................... 40 Cas9 Effector Expression ................................................................................................... 40 Location, Location, Location ............................................................................................. 41 System Selection ................................................................................................................ 41 Additional Applications of CRISPR-Cas Systems .................................................................. 42 Plasmid Curing ................................................................................................................... 42 CRISPR-based “Magic Bullets” ......................................................................................... 43 CRISPR-based Memory Devices ....................................................................................... 46 CRISPR-based Fluorescent Imaging .................................................................................. 46 In Vitro CRISPR-based Applications ................................................................................. 48 iii RNA Targeting ................................................................................................................... 49 Conclusions ............................................................................................................................. 49 Acknowledgments ................................................................................................................... 51 References ............................................................................................................................... 51 CHAPTER 3. A MODULAR CRISPR INTERFERENCE GENETIC TOOLKIT FOR CONDITIONAL GENE REPRESSION IN ESCHERICHIA COLI ............................................68 Abstract .................................................................................................................................... 68 Introduction ............................................................................................................................. 69 Materials and Methods ............................................................................................................ 71 Strains and Materials .......................................................................................................... 71 Genetic Constructions ........................................................................................................ 76 dCas9 Vector Integration.................................................................................................... 77 Targeted Repression of lac Operon Genes and β-Galactosidase Assays ........................... 78 Antibiotic Disc-diffusion Assays ....................................................................................... 79 Gene Essentiality Growth Curve Assays............................................................................ 79 Fluorescent Microscopy of Sec Substrates during CRISPRi Repression........................... 79 Translocation Pathway Depletion Assays .......................................................................... 80 Combinatorial CRISPRi Targeting of the lac Operon and Essential Gene metG .............. 80 Results ..................................................................................................................................... 81 Efficient Protocol for Integration of dCas9 ........................................................................ 81 Repression of the lac Operon ............................................................................................. 82 CRISPRi-mediated Antibiotic Sensitivity .......................................................................... 83 Essential Gene Repression with CRISPRi ......................................................................... 84 Characterizing Translocation-pathway Determinants of Membrane Proteins Using CRISPRi ............................................................................................................................