Engineering Cre Recombinase for Genome Engineering Chi Zhang Washington University in St

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Engineering Cre Recombinase for Genome Engineering Chi Zhang Washington University in St Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Spring 5-15-2016 Engineering Cre Recombinase for Genome Engineering Chi Zhang Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Biology Commons Recommended Citation Zhang, Chi, "Engineering Cre Recombinase for Genome Engineering" (2016). Arts & Sciences Electronic Theses and Dissertations. 758. https://openscholarship.wustl.edu/art_sci_etds/758 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Computational and Systems Biology Dissertation Examination Committee: James Havranek, Chair Joseph Corbo Robi Mitra Gary Stormo Ting Wang Engineering Cre Recombinase for Genome Editing by Chi Zhang A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2016 St. Louis, Missouri © 2016, Chi Zhang Table of contents List of Figures ................................................................................................................................. v List of Tables ................................................................................................................................. vi Acknowledgments ........................................................................................................................ vii ABSTRACT OF THE DISSERTATION ...................................................................................... ix Chapter 1 Introduction: Directed Evolution, Cre Recombinase and RMCE .................................. 1 Directed evolution of enzymes .................................................................................................... 2 The Cre-lox system ..................................................................................................................... 4 Altering Cre’s DNA specificities ................................................................................................ 4 Symmetry of Cre recombinase tetramer...................................................................................... 7 Recombination mediated cassette exchange (RMCE) ................................................................ 7 Advantages of RMCE over double-stranded break (DSB) stimulated homology-directed repair (HDR) .......................................................................................................................................... 9 Focus of the dissertation ............................................................................................................ 11 References ................................................................................................................................. 12 Chapter 2 Redesign of the Monomer-monomer Interface of Cre Recombinase Yields an Obligate Heterotetrameric Complex ............................................................................................. 21 ABSTRACT .............................................................................................................................. 22 INTRODUCTION ..................................................................................................................... 22 MATERIALS AND METHODS .............................................................................................. 26 Computational modeling and design ................................................................................ 26 Gene construction and protein expression ........................................................................ 26 In vitro recombinase activity assay ................................................................................... 27 Cell culture and transfection ............................................................................................. 27 Flow cytometry ................................................................................................................. 29 Recombinase assay in mouse retinal explants .................................................................. 29 RESULTS.................................................................................................................................. 30 Computational redesign of a non-native but functional protein-protein interface between Cre recombinase monomers .............................................................................................. 30 ii Iterative rounds of rational design enhance the formation of (ABAB) complexes .......... 31 Heterotetrameric mutations can be combined with DNA specificity altering mutations to enhance target site specificity ........................................................................................... 33 Obligate heterotetramer formation is preserved in mammalian cells ............................... 34 DISCUSSION ........................................................................................................................... 35 FUNDING ................................................................................................................................. 40 REFERENCES .......................................................................................................................... 41 Chapter 3 Rec-Seq: A High-Throughput Specificity Assay for Recombinases ........................... 58 ABSTRACT .............................................................................................................................. 59 INTRODUCTION ..................................................................................................................... 59 MATERIALS AND METHODS .............................................................................................. 63 Molecular cloning, expression, and purification of recombinases ................................... 63 Library preparation for Illumina sequencing .................................................................... 64 High-throughput sequencing data analysis ....................................................................... 64 Identification of Cre homologs and prediction of their RT sites ...................................... 65 RESULTS.................................................................................................................................. 66 A high-throughput assay of recombinase DNA specificity .............................................. 66 NaCl concentration has a large effect on observed in vitro specificity ............................ 67 Sequence specificity for Cre variants and homologs ........................................................ 68 Probing the sequence specificity of an evolved recombinase ........................................... 70 DISCUSSION ........................................................................................................................... 71 REFERENCES .......................................................................................................................... 74 Chapter 4 Conclusions and Future Directions ............................................................................. 90 Design of recombinases for asymmetric sites ........................................................................... 91 Prospects for additional heterotetrameric recombinase pairs .................................................... 91 Rec-Seq is a powerful tool to study the DNA specificity of recombinases .............................. 93 Accelerating the retargeting of recombinases ........................................................................... 94 References ................................................................................................................................. 95 iii Appendix I Supplemental Materials for Chapter 2 ....................................................................... 97 Supplemental Methods .............................................................................................................. 98 References ................................................................................................................................. 98 Appendix II Supplemental Materials for Chapter 3 .................................................................... 103 Primers for library generation (See Supplemental Table 1 for RT site sequence) .................. 107 Primers for Illumina sequencing preparation .......................................................................... 107 iv List of Figures Figure 1.1 34 bp loxP DNA sequence .......................................................................................... 18 Figure 1.2 Synaptic complex of Cre homotetramer ...................................................................... 19 Figure 1.3 Schemes of RMCE ...................................................................................................... 20 Figure 2.1. Genomic applications of Cre recombinase. ................................................................ 48 Figure 2.2. Mutated positions in
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