Engineering and Characterization of Cas9 Nuclease

dc.contributor.advisorZhang, Xiaoliu Shaun
dc.contributor.committeeMemberChung, Sang-Hyuk
dc.contributor.committeeMemberFrigo, Daniel E.
dc.contributor.committeeMemberCirino, Patrick C.
dc.contributor.committeeMemberLin, Chin-Yo
dc.creatorSpencer, Jeffrey Michael 1986-
dc.creator.orcid0000-0003-4690-2432
dc.date.accessioned2018-03-12T17:58:59Z
dc.date.available2018-03-12T17:58:59Z
dc.date.createdDecember 2017
dc.date.issued2017-12
dc.date.submittedDecember 2017
dc.date.updated2018-03-12T17:58:59Z
dc.description.abstractThe widespread use of CRISPR-associated nucleases has invigorated the field of site-specific nucleases. The success of Streptococcus pyogenes Cas9 (SpCas9) has led to the discovery of several other CRISPR-associated nucleases. As more CRISPR-associated nucleases become available, it will be necessary to understand and refine their activity before they can be translated into the clinic. With this in mind, my dissertation demonstrates the utility of characterizing unique Cas9 orthologs, using Neisseria meningitidis Cas9 (NmCas9) as a model. Additionally, we sought to demonstrate how deep mutational scanning (DMS) could provide details about important functional regions in SpCas9 and speed its engineering. Chapter 1 discusses the unique properties of NmCas9. We define a highly active gRNA structure that will allow the use of NmCas9 in biotechnology. We found that SpCas9 fails to effectively engage DNA using this gRNA. Next, we looked at the properties of NmCas9’s protospacer adjacent motif (PAM). Divergent from SpCas9, NmCas9’s PAM tolerates several different nucleotide mutations, providing flexibility in the sequences that can be targeted by this ortholog. NmCas9 also has the ability to discriminate between perfectly matched sequences and those with two mismatches. These features will allow NmCas9 to be a useful tool in genome editing applications. Chapter 2 focuses on the results of DMS experiments aimed at elucidating important features of SpCas9. In this work, we developed a nuclease screening platform which could distinguish active Cas9 mutants. We screened a library of 1.7 x 107 with over 8500 possible non-synonymous mutations and inferred the effects of each mutation using DMS. We demonstrate that the RuvC and HNH domains are the least tolerant regions to mutation. In contrast, the Rec2 and PI domains tolerate mutation better than other regions. The mutation information defined in this work provides a foundation for further SpCas9 engineering. Our results demonstrate how DMS can be a powerful tool to uncover features important to CRISPR-associated nuclease function. Application of this approach to emerging CRISPR-associated nucleases should enhance their engineering and optimization for therapeutic and other applications.
dc.description.departmentBiology and Biochemistry, Department of
dc.format.digitalOriginborn digital
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/10657/2855
dc.language.isoeng
dc.rightsThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).
dc.subjectCas9 CRISPR Nuclease
dc.titleEngineering and Characterization of Cas9 Nuclease
dc.type.dcmiText
dc.type.genreThesis
local.embargo.lift2019-12-01
local.embargo.terms2019-12-01
thesis.degree.collegeCollege of Natural Sciences and Mathematics
thesis.degree.departmentBiology and Biochemistry, Department of
thesis.degree.disciplineCell and Molecular Biology
thesis.degree.grantorUniversity of Houston
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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