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CHARACTERIZING THE EFFECT OF CRISPR/CAS9 ON BASE EXCISION REPAIR
Dissertation   Open access

CHARACTERIZING THE EFFECT OF CRISPR/CAS9 ON BASE EXCISION REPAIR

Jacob S. Antony
Washington State University
Doctor of Philosophy (PhD), Washington State University
05/2024
DOI:
https://doi.org/10.7273/000006481
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Abstract

CRISPR/Cas9 In vitro R-loops Yeast DNA Repair Genetics Mutagenesis
While genetic material is relatively stable it is mutable and accumulates DNA damage from many endogenous and exogenous sources. DNA repair pathways are required to remove that damage and prevent genome instability resulting from unrepaired damage. Importantly, mutations generated following the repair of damage induced by CRISPR/Cas9 is critical for traditional genome editing approaches. However, many CRISPR-based technologies rely on catalytically dead Cas9 (dCas9) which only binds DNA. Given that binding alone can be mutagenic, the work presented in this dissertation focuses on further understanding the mechanisms in which binding alone induces mutations. We first examined whether the targeting of dCas9 inhibits the repair of uracil and inosine base lesions through base excision repair (BER) in vitro. This led to the discovery that dCas9 is inhibitory to multiple classes of DNA glycosylases in vitro, likely through steric occlusion (i.e., preventing access to lesions). Moreover, our data implies that the mechanisms DNA glycosylases employ to bind and remove damaged DNA likely impact how dCas9 impairs their activity. We also addressed whether inhibition of repair occurs in cells by investigating how exposure to endogenous mutagen like methylmethanesulfonate (MMS) impact dCas9-associated mutagenesis in the yeast Saccharomyces cerevisiae. Interestingly, we saw that in cells expressing both dCas9 and guideRNAs targeting the CAN1 gene in yeast, MMS exposure significantly increased both mutation frequencies and rates in repair-proficient cells. We speculate that there could be multiple mechanisms driving this mutagenesis and that persistent binding by Cas9 (or dCas9) likely promotes background mutations arising during genome editing.

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