In Vivo Visualization of mRNA-Driven Base Editing Activity in Tumours

Krautner, Joshua Stefan Tatsuo

In Vivo Visualization of mRNA-Driven Base Editing Activity in Tumours

Files

Krautner_Joshua_MSc_2025_Thesis.pdf (2.23 MB)

Date

2025-10-30

Authors

Krautner, Joshua Stefan Tatsuo

Abstract

CRISPR technologies are transforming the treatment of diseases such as cancer by enabling precise genome editing. These technologies use a guide RNA to direct a CRISPR-associated protein to a specific DNA sequence, allowing targeted gene knockouts or knock-ins. However, early CRISPR approaches relied on generating double-strand breaks (DSBs), which can lead to unpredictable and potentially harmful consequences. To address these risks, base editors (BEs) were developed. BEs enable precise single-nucleotide changes without introducing DSBs, offering a safer alternative for therapeutic applications.

At present, BE activity is typically validated through ex vivo DNA sequencing, which requires invasive sampling and cannot track editing dynamics in living systems. Molecular imaging offers a complementary strategy by providing non-invasive, longitudinal information about biological processes within the same animal or patient. This thesis investigates an activatable bioluminescence “gene-on” (GO) reporter system, Akaluc GO, to dynamically visualize BE activity in vivo. Using liposome-mediated intratumoural delivery of mRNA-encoded BEs, we demonstrate the potential of this system to serve as a non-invasive tool for monitoring base editing and advancing CRISPR therapeutic development.

Summary for Lay Audience

Cancer remains one of the leading causes of death, and while treatments such as chemotherapy, radiotherapy, and immunotherapy have improved outcomes, cancer remains difficult to treat. A promising new strategy is genome editing, which enables the direct alteration of DNA inside cells. By precisely reprogramming the genetic changes that drive cancer, genome editing holds the potential to generate more targeted and effective therapies. Clustered regularly spaced short palindromic repeat (CRISPR)/Cas9, first discovered in bacteria as a defense against viruses, was adapted in 2012 into a powerful tool for modifying the genome of human cells. Initial CRISPR systems used RNA to direct a CRISPR-associated protein to a specific sequence to knockout or knock-in genes. More recently, next-generation technologies have expanded the ability to manipulate genomes with greater precision and potentially improved safety. Among these, base editors enable the direct conversion of one DNA base pair into another, offering a way to correct or disrupt disease-causing mutations. Currently, these CRISPR technologies are being developed for treating diseases such as cystic fibrosis, muscular dystrophy, as well as cancer. Despite this promise, clinical translation faces significant hurdles, including the safe and efficient delivery of CRISPR components, minimizing unintended DNA or RNA alterations, and controlling immune responses to the bacterial CRISPR/Cas9 proteins. These safety concerns highlight the need to monitor genome editing outcomes in both preclinical models and patients. Determining whether editing has occurred inside living organisms is a crucial step for assessing CRISPR technologies, often requiring invasive biopsies that limit the ability to monitor biological changes over time. To address this, reporter systems have been developed to function like biological “light switches”. In this project, a bioluminescent reporter gene called Akaluc was engineered with a mutation that causes expression to remain “off” until base edited. When Akaluc switches “on”, base edited cells emit light that can be detected in real time. This thesis establishes a non-invasive ‘gene on’ (GO) bioluminescent reporter system, Akaluc GO, as a tool to monitor base editing in cancer models. By enabling real-time visualization of editing activity in tumours, AkalucGO provides a valuable method to guide the safe development of future genome editing technologies for cancer therapies.