An Exploration of the Properties of Repair Template DNA that Promote Precision Genome Editing
Authors
Ghanta, Krishna S.Faculty Advisor
Craig MelloAcademic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
RNA Therapeutics InstituteDocument Type
Doctoral DissertationPublication Date
2021-08-03Keywords
C. elegansCRISPR
Genome editing
HDR
Donor DNA
Precision genome editing
Chemical Modifications
Modified donors
5′ modifications
TEG modifications
melted donors.
Biotechnology
Genetics
Molecular Biology
Molecular Genetics
Metadata
Show full item recordAbstract
CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms. In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing. Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.DOI
10.13028/hs7v-w341Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31382Rights
Licensed under a Creative Commons licenseDistribution License
http://creativecommons.org/licenses/by/4.0/ae974a485f413a2113503eed53cd6c53
10.13028/hs7v-w341
Scopus Count
Except where otherwise noted, this item's license is described as Licensed under a Creative Commons license