Activating Endogenous Reporter Genes to Visualize CRISPR-activation (CRISPRa) Based Therapies

Transcript

Hello, my name is Emanuel Silvester, and I am a senior at London Central Secondary School. Inspired by my co-op experience in molecular imaging, my project addresses a critical gap in CRISPR technology. While CRISPR is a groundbreaking, tunable tool for gene activation, its in vivo use is limited by the inability to monitor its activity without invasive biopsies or tissue staining.

To solve this, I utilized a catalytically inactive dCas9-VPR system to activate the Sodium Iodide Symporter (also known as NIS), a PET imaging reporter gene. By testing in vitro various guide RNAs, I demonstrated that dCas9-VPR is a robust, tunable CRISPRa system capable of inducing NIS mRNA expression in HeLa cells. This research paves the way for non-invasively tracking gene therapies, for future work in ensuring CRISPRa systems reach their targets safely and effectively.

Intro

In 2012, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system were repurposed from the bacterial adaptive immune system for genome editing (Jinek et al., 2012). While initial iterations relied on a guide RNA (gRNA) to direct Cas9 to induce double-strand breaks at complementary DNA sequences, subsequent innovations have expanded the technology's utility beyond simple genome editing. In 2013, the development of CRISPR activation (CRISPRa) enabled the targeted upregulation of endogenous genes without altering the underlying DNA sequence. These systems utilize catalytically inactive "dead" Cas9 (dCas9) fused to potent transcriptional activators, such as VP64-p65-Rta (VPR) to recruit the cellular transcriptional machinery directly to a target locus (Chavez et al., 2015) (Fig. 1).

However, a major limitation for in vivo CRISPRa applications is monitoring delivery and activity of the CRISPRa systems. Current verification methods often rely on invasive biopsies in patients or terminal tissue staining in animal studies. To address this, the sodium iodide symporter (NIS) serves as a promising endogenous reporter utilized in positron emission tomography (PET) imaging. NIS expression is naturally low in most healthy tissues and many cancers, so its targeted activation offers an opportunity for non-invasive monitoring of CRISPRa systems (Yaghoubi et al., 2021) (Fig. 2).

Objective

Evaluated a CRISPRa system called dCas9-VPR system to activate the expression of the endogenous sodium iodide symporter (NIS) gene, which is a positron emission tomography (PET) reporter gene.

Methods

HeLa cells were previously engineered to stably express dTomato fluorescence protein with dCas9-VPR (Fig 3.A). A fluorescence image post transduction was used to confirm the expression dTomato/dCas9-VPR construct (Fig. 3.B).

Experiments were performed in six steps (Fig. 4):

Guide RNAs (gRNAs) were encapsulated in liposomes via pipetting using the JetMessenger lipid reagent (Polyplus).

Liposomes were added to HeLa cells to enable the targeting of the dCas9-VPR system to the NIS gene.

At 24 - 168 hours post transfection total RNA was collected, including NIS messenger RNA (mRNA) and GAPDH mRNA.

RNA was used to synthesize complementary DNA (cDNA).

TaqMan probe kits for NIS/GAPDH (housekeeping gene) and cDNA were used to perform quantitative polymerase chain reaction (qPCR) using QuantStudio5 (Thermofisher).

Design & analysis tool 2.0 (Thermofisher) calculated relative quantification (RQ) of NIS (2-𝚫𝚫Ct) from raw Cycle threshold (Ct) values.

First, gRNA 3 was designed using CRISPick (Broad Institute) for initial pilot studies. Subsequently, gRNAs 1, 2, and 4, were designed by horizon Discovery. All gRNAs were synthesized from GenScript (Fig. 5).

24 hour dose-response experiment of gRNA 3

HeLa cells stably expressing dCas9-VPR (105 cells seeded overnight) were transfected with either non-targeted scrambled gRNA control or NIS-targeted gRNA 3 at 0.25, 1, and 2 µg doses.

24 hour gRNA combinations experiment

HeLa cells expressing dCas9-VPR were transfected with 1 µg of gRNA using all possible combinations 1 through 4 at equal mass ratios.

Time course experiment with gRNA 1 + 3

HeLa cells expressing dCas9-VPR (5104 cells seeded overnight) were transfected with 1 µg of gRNA combination 1 + 3. Total RNA was collected for proceeding steps after 24, 48, 120, and 168 hours.

Results and Analysis

Confirming delivery of all components

Initial validation via gel electrophoresis confirmed NIS messenger RNA (mRNA) expression in transfected groups, while the non-transfected control (NTC) and scrambled (Scr) gRNA negative control exhibited no significant NIS amplification (Fig. 6).

24 hour dose-response experiment of gRNA 3

To determine the optimal amount of gRNA for activation, a dose-response experiment was conducted with doses (0.25, 1, and 2 µg) of gRNA 3 (Fig. 7). At 24 hours post-transfection, all doses of NIS-targeted gRNA showed significantly larger fold change of NIS mRNA expression relative to scrambled controls. Since the 2 µg dose resulted in a plateau, the 1 µg dose was used for subsequent experiments.

24 hour gRNA combinations experiment

Following dose optimization, a combination experiment was performed to determine the synergistic potential of pooling different gRNAs at equivalent mass ratio (total 1 µg) (Fig. 8). This screening revealed gRNA 1 + 3 produced the greatest increase in NIS mRNA expression, suggesting that spatial orientation relative to the transcription start site plays a significant role in dCas9-VPR recruitment of transcription machinery. While this combination provided the highest peak expression, combinations involving three gRNAs demonstrated greater stability across conditions.

Time course experiment with gRNA 1 + 3

A time-course analysis was performed using the gRNA 1 + 3 combination to assess the duration of NIS expression (Fig. 9). The peak NIS mRNA expression was observed at 24 hours post-transfection, followed by a statistically significant decrease after 48 hours. Notably, NIS expression levels remained significantly elevated and steady from 48 through 168 hours compared to the Scr control. This shows that while the initial peak subsided, the dCas9-VPR system maintained a sustained "on-state" for up to one week.

Summary

Demonstrated 1 µg dose is optimal for NIS mRNA activation in HeLa cells (24 well).

Combination of gRNA 1 + 3 yielded the highest NIS mRNA activation.

Peak mRNA expression after transfecting gRNA 1 + 3 occurred at 24 hours and remained stable up to one week.

Discussion

The development of CRISPR activation (CRISPRa) technologies remains a significant challenge. Currently, monitoring both delivery and functional activity in vivo relies on invasive biopsy or end point tissue staining, leaving a critical gap in our ability to track both outcomes in real-time. To address this unmet need, the CRISPRa system, dCas9-VPR, was evaluated for its ability to induce an endogenous reporter called sodium iodide symporter (NIS) gene in HeLa cells. NIS serves as a functional imaging reporter by mediating the cellular uptake of PET radiotracers. The successful induction of NIS mRNA expression, translation, and relocation to the cell membrane provides a framework for both non-invasive longitudinal monitoring of delivery and activity. Such tools have the potential for accelerating preclinical evaluation and support clinical translation of CRISPRa therapies.

To assess when NIS mRNA expression peaks, a time-course experiment was performed using the gRNA 1 + 3 combination as it had the greatest synergistic effect among all other gRNA combinations. After 24 hours, NIS expression peaked and remained significantly elevated compared to Scr controls for over seven days. Interestingly, the variability observed between different gRNA combinations suggests that activation is influenced by the precise positioning of the gRNA relative to the transcription start site (TSS) and the physical spacing between dCas9-VPR complexes (Beyersdorf et al., 2022).

Takeaway

The application of the CRISPRa system dCas9-VPR for activating NIS mRNA expression potentially provides the reporter gene expression necessary for longitudinal PET imaging, holding promise as a tool for guiding CRISPRa-based therapeutics.

Limitations

NIS protein expression, membrane localization, and PET radiotracer uptake have not yet been assessed.

dCas9-VPR NIS activation in HeLa cells may not generalize across different cell lines, gRNA targets, CRISPRa systems, or in vivo contexts.

Future Work

Assess functional NIS activity through iodine uptake.

Test the dCas9-VPR system in vivo in tumours through PET imaging after liposome delivery of gRNA.

Extend work to other cell lines including healthy cells.

First, I would like to thank the members of the Cellular and Molecular Imaging Group at Robarts Research Institute including Dr. John A. Ronald, PhD to whom I owe this project idea, and Joshua Krautner, MSc for his unending support and mentorship. I would also like to thank the Thames Valley Science and Engineering Fair Regional Coordinator and delegates for their peer-editing work and helpful suggestions for my ProjectBoard. Lastly, I would like to thank my parents for their endless support and encouragement throughout this project.

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