Precise and Expansive Genomic Positioning for CRISPR Edits MASSADHIUSET[NSTITUTE by JUL 2 6 2019 Noah Michael Jakimo L I LIBRARIES 19 B.S., California Institute of Technology (2010) S.M., Massachusetts Institute of Technology (2015) Submitted to the Program in Media Arts and Sciences, School of Architecture and Planning in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Media Arts and Sciences at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2019 ©Massachusetts Institute of Technology 2019. All rights reserved. Signature redacted A uthor ................................ Program in Medd Arts and Sciences, School of Architecture and Planning May 3, 2019 Certified by ... .. ....... Signature redacted Joseph M. Uacobson Associate Professor of Media Arts and Sciences Thesis Supervisor Accepted by ............. Signatureredacted (j)Tod Machover Academic Head, rogram in Media Arts and Sciences 77 Massachusetts Avenue Cambridge, MA 02139 MITLibraries http://Iibraries.mit.edu/ask DISCLAIMER NOTICE Due to the condition of the original material, there are unavoidable flaws in this reproduction. We have made every effort possible to provide you with the best copy available. Thank you. Some pages in the original document contain text that is illegible. t Precise and Expansive Genomic Positioning for CRISPR Edits by Noah Michael Jakimo Submitted to the Program in Media Arts and Sciences, School of Architecture and Planning on May 3, 2019, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Media Arts and Sciences Abstract The recent harnessing of microbial adaptive immune systems, known as CRISPR, has enabled genome-wide engineering across all domains of life. A new generation of gene-editing tools has been fashioned from the natural DNA/RNA-targeting ability of certain CRISPR-associated (Cas) proteins and their guide RNA, which work together to recognize and defend against infectious genetic threats. This straight-forward RNA-programmed sequence recognition by CRISPR has facilitated its rapid global impact on genetic research, diagnostics, therapeutics, and bioproduction. An ideal DNA-editing platform would achieve perfect accuracy on any desired cellular and genomic target. CRISPR systems, however, have limited target fidelity and range, in part due to their evolutionary pressures to defend microbes from fast- mutating viruses without self-targeting their own guide RNA. These natural limita- tions of CRISPR can especially constrain gene-editing in animals and plants, which are more vulnerable to off-target activity occurring in one of their trillions of cells with genomes that are 1000x larger than those of unicellular microbes that natively harbor CRISPR systems. This thesis overcomes three critical challenges for precise and broad gene-editing of complex organisms: 1) engineering a means of specificity for the type of cells to edit, 2) improving target-matching accuracy, and 3) broadening the editable portion of the genome. This thesis addresses these challenges by integrating custom developed computa- tional design tools and biological validation of the resulting novel CRISPR systems; 1) To target within multicellular heterogeneity, new oligonucleotide-sensing structural motifs are designed and embed into guides that can potentially control CRISPR nucle- ase activity based on cell-type transcriptome patterns; 2) To discern among increased similarity between a target and non-target sequences in larger genomes, base-pairing thermostability principles are employed to tune the biochemical composition of guides that can evade subtly mismatched off-target sites; 3) To expand the reach of editing techniques with narrow windows of operation, such as base-editing, bioinformatics workflows that discover previously uncharacterized Cas proteins with novel target scope are created. This thesis demonstrates the effectiveness of these strategies in the context of in vitro, bacterial, and human cell culture assays, and contributes advancements in the precision and generality for CRISPR gene-editing. Thesis Supervisor: Joseph M. Jacobson Title: Associate Professor of Media Arts and Sciences I s Precise and Expansive Genomic Positioning for CRISPR Edits by Noah Michael Jakimo The following people served as readers for this thesis: I Signature redacted Professor Joseph M. Jacobson........ Thesis Supervisor Associate Professor of Media Arts and Sciences, MIT S i jnaUIt 1rlU re d4 alezt d - Professor Edward S. Boyden III... Thesis Reader Associate Professor of Media Arts and Sciences, MIT -~ Signature redacted Professor George M. Church.. Thesis Reader Professor of Genetics, Harvard Medical School Dedication For Teri, who makes every day wonderful. We are lucky to have each other. t Acknowledgments My thesis was made possible by the kind and generous support of my advisor, mentors, labmates in Molecular Machines, co-workers at the MIT Media Lab (ML) and Center for Bits and Atoms (CBA), friends, family, and, of course, my wife, Teri. I would like to give special thanks: To Joe, for the opportunity to learn, invent, explore, and discover together; To Pranam, for our balanced and productive partnership; To Lisa and Thras, for cohesive team efforts through struggles and successes; To Neil and Joi, for the feeling of belonging in both the CBA and Media Lab; To CBA and ML, for being empowering environments for myself and the world; To George and Ed, for sharing your prolific genius and caring guidance; To CBA and ML admins, for enabling our capabilities and focus; To Linda, for overseeing our degree-seeking academic trajectory; To Naama and David, for vital experience and knowledge; To mom and dad, Benay and Alan, for a lifetime of loving encouragement; To Teri, for shared love and inspiration. From the bottom of my heart, thank you! Contents 1 CRISPR Systems for Gene Editing 15 1.1 Introduction ........... .... ... 16 1.2 Native CRISPR Mechanics ... ....... 16 1.3 CRISPR for Gene-Editing . ... .. .. 17 1.3.1 Cas9 Range ....... ... .... 19 1.3.2 Cas9 Specificity ... .. .. ... 20 1.3.3 Cas9 Activity . ..... ..... .. 21 1.4 Thesis Contributions ...... ... .. 22 2 DNA/RNA Chimeric CRISPR Guides Enhance Target Specificity for Streptococcus pyogenes Cas9 (SpCas9) 23 2.1 Introduction ............ ........ 24 2.2 Results and Discussion ..... ......... 25 2.2.1 DNA Substitutions in Cas9 gRNA Improve Mismatch Sensitivity 25 2.2.2 R-Loop Expansion Kinetics Determine Melt-Guide Specificity 28 2.2.3 Melt-Guides Reduce Off-Target Genome Editing ........ 29 2.2.4 Conclusions . ....... ........ ........ .... 32 2.3 Materials and Methods . ......... ........ ........ 32 2.3.1 Cas9-Guide in vitro DNA Digestions . ...... ...... 32 2.3.2 Preparation of Single-Stranded Target DNA Substrates ... 33 2.3.3 Genomic Indel Production and Measurements .... ..... 33 2.3.4 Sequence Information ... ...... ...... ...... .. 34 11 3 Single-Base PAM Specificity of a Highly-Similar SpCas9 Ortholog from Streptococcus canis 35 3.1 Introduction ........... ............. ........ 36 3.2 R esults .. ....... ...... ...... ...... ...... .. 37 3.2.1 Identification of SpCas9 Homologs ................ 37 3.2.2 Determination of PAM Sequences Recognized by ScCas9 .. 38 3.2.3 Assessment of ScCas9 PAM Specificity in Human Cells ... 39 3.2.4 Off-Target Analysis of ScCas9 ...... ........... 41 3.2.5 ScCas9 Genome Editing Capabilities .... .......... 43 3.2.6 Investigation of Sequence Conservation Between S. canis and Other Streptococcus Cas9 Orthologs ....... ........ 46 3.2.7 Genus-wide Prediction of Divergent Streptococcus Cas9 PAMs 48 3.3 D iscussion .. ........ ........ ......... ...... 51 3.4 M aterials and M ethods ... ............ ........... 54 3.4.1 Identification of Cas9 Homologs and Generation of Plasmids . 54 3.4.2 PAM-SCANR Assay ... ...... ...... ...... .. 55 3.4.3 Cell Culture and Gene Modification Analysis .. ...... .. 56 3.4.4 Base Editing Analysis with Traffic Light Reporter ... .... 57 3.4.5 Base Editing Evaluation Program .... ........ .... 57 3.4.6 SPAMALOT Pipeline ... ...... ...... ...... 58 3.4.7 Statistical Analysis .... ........ ........ .... 58 4 A Cas9 with Complete PAM Recognition for Adenine Dinucleotides from Streptococcus macacae 59 4.1 Introduction ... ...... ....... ...... ...... .... 60 4.2 M aterials and Methods .. ....... ...... ...... .... 69 4.2.1 Selection of Streptococcus Cas9 Orthologs of Interest . .... 69 4.2.2 PAM-SCANR Bacterial Fluorescence Assay ..... ..... 69 4.2.3 Purification of and DNA cleavage with Selected Nucleases . 70 4.2.4 Gene Modification Analysis and Software ...... ...... 72 12 4.2.5 Base Editing Analysis and Software .............. 72 5 RNA-Switched Cas9 Guides Engineered by Strand-Displacement 77 5.1 Introduction ................................ 78 5.2 Computational Design of Switchable Guide RNA ........... 78 5.3 Validation of the Toehold-Gated Strand-Displacement Mechanism . 81 5.4 Demonstration of OFF-to-ON and ON-to-OFF Switchable Guide RNA 82 5.5 M aterials and Methods ...... .................... 83 5.5.1 In vitro Cas9 Cleavage Assays .................. 83 5.5.2 Nupack swigRNA Design ..................... 84 6 Concluding Remarks 85 7 Bibliography 89 13 14 Chapter 1 CRISPR Systems for Gene Editing 15 '1 Growth in CRISPR and Gene Editing Publications Since 2010 5000- = "Gene Editing" in abstract -A-"TALEN" in abstract 4000 - "CRISPR" in abstract "CRISPR" 2-year doubling projection Uo 3000 - 0 .2000- 1000- 0- 2010 2011 2012 2013 2014 2015 2016 2017 2018 Figure 1.1 Crowth