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Swyersnathan.Pdf DEVELOPMENT OF AN AMENABLE SYSTEM FOR SITE-SPECIFIC ADDITION TO A MAIZE CHROMOSOME _______________________________________ A Dissertation presented to the Faculty of the Graduate School at the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _____________________________________________________ by NATHAN CHARLES SWYERS Dr. James A. Birchler, Dissertation Supervisor December, 2019 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled DEVELOPMENT OF AN AMENABLE SYSTEM FOR SITE-SPECIFIC ADDITION TO A MAIZE CHROMOSOME Presented by Nathan Charles Swyers, A candidate for the degree of Doctor of Philosophy, And hereby certify that, in their opinion, it is worthy of acceptance. Dr. James A. Birchler Dr. Kathleen Newton Dr. David Braun Dr. Sherry Flint-Garcia ACKNOWLEDGMENTS The following list of people helped make this work possible: Birchler Lab: MU Biological Sciences: • Rebecca Ballew • Patrice Albert • David Braun • James A. Birchler • Lori Eggert • Weihong Chen • Nila Emerich • Robert Gaeta • Benjamin Julius • Rick Masonbrink • Melody Kroll • Ryan Donohue • Kathleen Newton • Ryan Douglas • Will Swatson • Lin Sun Family • Adam Johnson • Amie Swyers • Zhi Gao • Gideon Swyers • Nathaniel Graham • Michael Swyers • Becca Lukasak • Brenda Swyers • Morgan McCaw • Charles Swyers • Xiaowen Shi • Rita Henderson • Jon Cody • Michael Noble • Hua Yang • Jill Noble • Changzeng Zhao • Robert Fritz • Adam Gray Friends • Alana Rodney • Seth Nelson • Leesa Nelson • John Bauer ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ II LIST OF FIGURES .......................................................................................................... VI LIST OF TABLES ............................................................................................................ IX ABSTRACT ........................................................................................................................ X CHAPTER 1: 1. DEVELOPMENT OF AN AMENDABLE SYSTEM FOR SITE-SPECIFIC ADDTION TO A MAIZE CHROMOSOME .........................................................1 Summary ................................................................................................................ 2 Introduction ............................................................................................................ 2 Platform for Genome Engineering ....................................................... 4 Centromeres ......................................................................................... 5 Telomeres............................................................................................. 6 Origins of Replication .......................................................................... 6 Artificial Chromosomes ....................................................................... 7 Telomere Mediated Chromosomal Truncation .................................... 8 B Chromosomes ................................................................................... 9 Recombinases .................................................................................... 13 Cre Recombinase ............................................................................... 17 Phi C31 Integrase ............................................................................... 18 FLP Recombinase .............................................................................. 18 Project Overview ............................................................................... 19 Truncation Construct pNT1 ............................................................... 23 Amendment Construct 1 .................................................................... 25 Amendment Construct 2 .................................................................... 25 Materials and Methods ......................................................................................... 26 Construct Design ................................................................................ 26 Transformation ................................................................................... 41 Screening Transformants ................................................................... 43 Results .................................................................................................................. 46 iii pPZPGLP1 Alternative ...................................................................... 62 Discussion ............................................................................................................ 66 Future Directions ................................................................................................. 67 AC1FRT, AC2FRT ............................................................................ 67 Cas9 B chromosome .......................................................................... 69 Pollen Selection Using apt1 ............................................................... 69 Baby boom and Wuschel Amendment Construct .............................. 70 AC1Telo............................................................................................. 73 References ............................................................................................................ 77 iv APPENDIX: 1. AMENDMENT CONSTRUCT MAPS ..................................................................86 2. TELOMERE-MEDIATED CHROMOSOMAL TRUNCATION FOR GENERATING ENGINEERED MINICHROMOSOMES IN MAIZE ..........101 3. PREPARATION OF CHROMOSOMES FROM ZEA MAYS ..............................134 4. FLUORESCENT IN SITU HYBRIDIZATION OF MAIZE (ZEA MAYS) CHROMOSOMES ...........................................................................................156 5. SEQUENCES OF PNT1, PPZPGLP1, AC1E, AND AC2E .................................200 VITA: ..............................................................................................................................229 v LIST OF FIGURES CHAPTER 1: FIGURE 1: B CHROMOSOME IN HI II BACKGROUND .....................................11 FIGURE 2: RECOMBINASE EXPRESSION CASSETTES....................................16 FIGURE 3: RECOMBINASE RECOMBINATION PRODUCTS ...........................19 FIGURE 4: PROPOSED AMENABLE SYSTEM ....................................................22 FIGURE 5: PNT1 AND PZPGLP1 CONSTRUCT MAPS .......................................24 FIGURE 6: PNT1 TELOMERE CLONES ................................................................29 FIGURE 7: SEQUENCING OF PNT1 CONSTRUCT .............................................30 FIGURE 8: AMENDMENT CONSTRUCT 1 ...........................................................32 FIGURE 9: AMENDMENT CONSTRUCT 2 ...........................................................33 FIGURE 10: AMENDMENT CONSTRUCTS WITH FRT SITES ..........................38 FIGURE 11: MAIZE TRANSFORMATION METHODS ........................................40 FIGURE 12: PNT1 INSERTED INTO B CHROMOSOME .....................................47 FIGURE 13: INTEGRITY CHECK OF PNT1 11 .....................................................49 FIGURE 14: SANGER SEQUENCING OF PNT1 11 ..............................................50 FIGURE 15: CRE-RECOMBINASE SELECTION REMOVAL .............................52 FIGURE 16: PNT1/CRE GEL ...................................................................................53 FIGURE 17: FISH IMAGE NEGATIVE CONTROL OF PNT1/CRE .....................55 FIGURE 18: PNT1/CRE FISH IMAGE ....................................................................56 FIGURE 19: SEQUENCING OF CRE-REDUCED PNT1 .......................................58 FIGURE 20: SEQUENCING OF CRE-REDUCED PZPGLP1 ................................59 FIGURE 21: PZPGLP1 TARGETING EXPERIMENT COMPONENTS ................61 FIGURE 22: PZPGLP1 ATTBDSRED AMENDMENT ..........................................64 FIGURE 23: PZPGLP1 AC1DSRED AMENDMENT .............................................65 vi FIGURE 24: STRATEGY FOR USE OF BBM AND WUS2 ...................................72 FIGURE 25: AC1TELOMERE POSSIBILITIES .....................................................74 SUPPLEMENTAL FIGURES SUPPLEMENTAL FIGURE 1: ATTBDSRED EXPERIMENT IMAGE ................76 APPENDIX 1: FIGURE 1: AC1E ......................................................................................................86 FIGURE 2: AC2E ......................................................................................................87 FIGURE 3: AC1GUS .................................................................................................88 FIGURE 4: AC1DSRED ............................................................................................89 FIGURE 5: AC1PHIC31INTEGRASE......................................................................90 FIGURE 6: AC1CAS9 ...............................................................................................91 FIGURE 7: AC1TELO ...............................................................................................92 FIGURE 8: AC1APT1 ...............................................................................................93 FIGURE 9: AC2DSRED ............................................................................................94 FIGURE 10: AC2PHIC31INTEGRASE....................................................................95
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