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dc.contributor.authorBessen, Jeffrey L.
dc.date.accessioned2019-12-12T09:03:30Z
dash.embargo.terms2021-05-01
dc.date.created2019-05
dc.date.issued2019-04-29
dc.date.submitted2019
dc.identifier.citationBessen, Jeffrey L. 2019. DNA Recombinases as Genome Editing Tools. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:42029711*
dc.description.abstractSite-specific recombinases (SSRs) have the potential to serve as ideal genome editing agents because they catalyze precise and efficient DNA strand exchange, but their innate specificity limits their applicability to a narrow range of DNA sequences. I have investigated several paths toward developing SSRs as viable genome editing tools. First, I describe the laboratory evolution of ROSACre, a variant of Cre recombinase that recognizes a human genomic target, using phage-assisted continuous evolution (PACE). We developed a PACE selection for recombinases and used it to evolve Cre to target a sequence in a genomic safe harbor. We demonstrated that ROSACre variants possess activity in mammalian cells on a target identical to a sequence within the human ROSA26 locus. Subsequently, I describe several alternative strategies, including adaptations of PACE as well as independent selections, in efforts to improve the activity and specificity of the resulting enzyme variants. Next I describe the development recCas9, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive Cas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA (gRNA) specified sequences. We show that recCas9 can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the gRNA sequences. DNA sequencing reveals that recCas9 catalyzes gRNA-dependent recombination in human cells with efficiency as high as 32% on plasmid substrates. Finally, we demonstrated that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Additionally, I describe efforts to improve the first-generation recCas9 construct by fusion of alternative recombinase domains. The engineering or evolution of SSRs into more versatile genome editing agents is limited in part by an incomplete understanding of SSR protein:DNA specificity determinants. To address this challenge, I describe the development of Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of evolved Cre variants Tre and Brec1, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells.
dc.description.sponsorshipChemistry and Chemical Biology
dc.format.mimetypeapplication/pdf
dc.language.isoen
dash.licenseLAA
dc.subjectsite-specific recombinases
dc.subjectgenome editing
dc.subjectprotein evolution
dc.subjectCre recombinase
dc.titleDNA Recombinases as Genome Editing Tools
dc.typeThesis or Dissertation
dash.depositing.authorBessen, Jeffrey L.
dash.embargo.until2021-05-01
dc.date.available2019-12-12T09:03:30Z
thesis.degree.date2019
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.grantorGraduate School of Arts & Sciences
thesis.degree.levelDoctoral
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
thesis.degree.nameDoctor of Philosophy
dc.contributor.committeeMemberLiu, David R.
dc.contributor.committeeMemberKahne, Daniel
dc.contributor.committeeMemberJoung, J. Keith
dc.type.materialtext
thesis.degree.departmentChemistry and Chemical Biology
thesis.degree.departmentChemistry and Chemical Biology
dash.identifier.vireo
dc.identifier.orcid0000-0002-0351-7393
dash.author.emailjeffbessen@gmail.com


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