VILNIUS UNIVERSITY Giedrius Gasiūnas Mechanism of DNA interference by Type II CRISPR/Cas systems Doctoral dissertation Physical science, biochemistry (04 P) Vilnius, 2012 The work presented in this doctoral dissertation has been carried out at the Institute of Biotechnology, Vilnius University during 2007-2011. Supervisor Prof. dr. Virginijus Šikšnys (Vilnius University, physical sciences, biochemistry - 04 P) 2 VILNIAUS UNIVERSITETAS Giedrius Gasiūnas DNR interferencijos mechanizmas II tipo CRISPR/Cas sistemose Daktaro disertacija Fiziniai mokslai, biochemija (04 P) Vilnius, 2012 3 Disertacija rengta 2007-2011 m. Vilniaus universiteto Biotechnologijos institute. Mokslinis vadovas Prof. dr. Virginijus Šikšnys (Vilniaus universitetas, fiziniai mokslai, biochemija - 04 P) 4 CONTENTS CONTENTS ...................................................................................................... 5 LIST OF ABBREVIATIONS .......................................................................... 8 INTRODUCTION ............................................................................................ 9 1. LITERATURE OVERVIEW ................................................................ 13 1.1. Bacterial antiviral defense mechanisms ................................... 13 1.2. CRISPR/Cas systems .................................................................. 16 1.3. Diversity and nomenclature of CRISPR/Cas systems and Cas proteins ........................................................................................ 19 1.4. Mechanisms of CRISPR/Cas systems ....................................... 23 1.4.1. Immunization step .................................................................... 23 1.4.2. Immunity step ........................................................................... 24 1.4.2.1. Generation of crRNA ........................................................ 26 1.4.2.2. crRNA guided interference ............................................... 26 1.5. Biochemistry of Cas proteins ..................................................... 27 1.5.1. Proteins involved in spacer acquisition .................................... 29 1.5.1.1. Cas1 protein ...................................................................... 29 1.5.1.2. Cas2 protein ...................................................................... 30 1.5.1.3. Csn2 protein ...................................................................... 31 1.5.2. Cas proteins involved in crRNA maturation ............................ 32 1.5.2.1. Cas6 protein family........................................................... 32 1.5.3. Complexes and Cas proteins involved in foreign nucleic acid recognition and degradation ..................................................... 35 1.5.3.1. Subtype II-E effector complex: Cascade .......................... 35 1.5.3.2. Cas3 .................................................................................. 40 1.5.3.3. Subtype I-F effector complex: Csy complex .................... 41 1.5.3.4. Type I-A effector complex: aCascade .............................. 42 1.5.3.5. The Cmr complexes .......................................................... 44 2. MATERIALS AND METHODS ........................................................... 49 2.1. Materials ...................................................................................... 49 2.1.1. Chemicals.................................................................................. 49 2.1.2. Enzymes .................................................................................... 49 2.1.3. Kits for molecular biology ........................................................ 49 2.1.4. Bacterial and viral strains ......................................................... 49 2.1.5. Plasmids and DNA ................................................................... 50 2.1.6. Oligonucleotide substrates ........................................................ 50 2.1.7. Buffers ...................................................................................... 50 2.2. Methods........................................................................................ 52 2.2.1. Electrophoresis ......................................................................... 52 2.2.1.1. Non-denaturing electrophoresis through agarose ............. 52 2.2.1.2. Non-denaturing polyacrylamide gel electrophoresis ........ 52 5 2.2.1.3. Denaturing polyacrylamide gel electrophoresis ............... 53 2.2.1.4. Denaturing (SDS) polyacrylamide gel electrophoresis of proteins ............................................................................. 53 2.2.2. DNA manipulations .................................................................. 54 2.2.2.1. Isolation of DNA and recombinant DNA techniques ....... 54 2.2.2.2. Construction of plasmid substrates ................................... 54 2.2.2.3. Cloning of full CRISPR3/Cas system .............................. 55 2.2.2.4. Frameshift mutations/deletions of cas genes .................... 55 2.2.2.5. Cloning of cas9 gene ........................................................ 57 2.2.2.6. Manipulations with CRISPR region ................................. 58 2.2.2.7. Site-directed mutagenesis ................................................. 60 2.2.3. Preparation of RNA substrates ................................................. 61 2.2.4. Plasmid transformation ............................................................. 61 2.2.5. Phage plaque assay ................................................................... 62 2.2.6. Expression and purification of Cas9 protein and Cas9-crRNA complex ..................................................................................... 62 2.2.7. Northern blot analysis ............................................................... 64 2.2.8. Reactions with oligonucleotide substrates ................................ 64 2.2.9. Reactions with plasmid substrates ............................................ 65 2.2.10. Plasmid cleavage position determination ............................. 65 2.2.11. Binding assay ........................................................................ 65 2.2.12. Sequence analysis ................................................................. 66 2.2.13. Gel filtration .......................................................................... 66 3. RESULTS ................................................................................................ 67 3.1. CRISPR/Cas systems of S. thermophilus .................................. 67 3.2. Characterization CRISPR3/Cas system in E. coli cells ........... 68 3.2.1. Cloning of the CRISPR3/Cas system in E. coli ........................ 68 3.2.2. The CRISPR3/Cas system prevents plasmid transformation in E. coli ........................................................................................ 70 3.2.3. The CRISPR3/Cas system provides resistance against phage lambda in E. coli ....................................................................... 71 3.2.4. Position-dependent tolerance for mismatches in CRISPR-based plasmid interference .................................................................. 72 3.2.5. The PAM sequence is important for plasmid interference ....... 73 3.2.6. Only Cas9 is required for plasmid interference in E. coli ........ 74 3.2.7. Mutational analysis of cas9 ...................................................... 75 3.2.8. The role of Cas proteins in the defense mechanism ................. 79 3.2.9. “Vaccination” of E. coli against plasmids and phages by the heterologous CRISPR3/Cas system .......................................... 80 3.3. The effector complex of Type II systems .................................. 81 3.3.1. Isolation and analysis of the Cas9-crRNA complex ................. 81 3.3.2. The Cas9-crRNA complex cleaves double-stranded DNA within the proto-spacer ........................................................................ 85 3.3.3. Cas9-crRNA cleavage specificity is directed by the crRNA sequence .................................................................................... 88 6 3.3.4. The PAM is required for DNA binding and cleavage by the Cas9-crRNA complex ............................................................... 92 3.3.5. Mutagenesis of RuvC and HNH motifs in Cas9 ....................... 94 3.3.6. The Cas9-crRNA complex guides dsDNA cleavage ................ 98 3.3.7. Mechanism of DNA interference in Type II systems ............... 99 3.3.8. Comparison to other effector complexes ................................ 101 LIST OF PUBLICATIONS ......................................................................... 105 CONFERENCE PRESENTATIONS .......................................................... 106 FINANCIAL SUPPORT .............................................................................. 107 ACKNOWLEDGEMENT ........................................................................... 108 REFERENCES .............................................................................................. 109 7 LIST OF ABBREVIATIONS aa amino acid Abi Abortive infection AHT anhydrotetracycline Ap ampycillin bp base pair BSA bovine serum albumin Cas CRISPR associated Cascade CRISPR-associated complex for antiviral defense Cm chloramphenicol CRISPR clustered regularly interspaced short palindromic repeats ds double-stranded DTT 1,4-dithiothreitol EDTA ethylenedinitrilotetraacetic acid EM electron microscopy EMSA electrophoretic mobility shift assay kb kilobase pair