Introduction: CRISPR systems are revolutionizing the treatment of disease at the genomic level. These technologies use a guide RNA (gRNA) to direct a CRISPR-associated (Cas) protein to a specific sequence to knockout or knock-in genes of interest¹. However, initial CRISPR systems cause double strand breaks that can lead to potentially harmful consequences², prompting the development of CRISPR 2.0 systems called base editors (BEs). BEs enzymatically convert one nucleotide to another without causing double strand breaks, providing opportunities to safely treat genomic diseases^3,4.

Currently, the outcomes of BE activity are validated through DNA sequencing technologies, which require invasive biopsies. An alternative would be to develop imaging tools that can indirectly provide spatiotemporal measures of in vivo BE activity. Previously, ‘Gene On’ (GO) fluorescence reporter systems were developed to visualize base edited cells in vitro⁵. Recently, a new GO system was developed using the highly sensitive bioluminescence imaging (BLI) reporter gene Akaluciferase⁶ (Akaluc^GO) to visualize editing following BE delivery with plasmid or minicircle DNA vectors (Persuasive Data file A and B)⁷. As seen with COVID-19 vaccines, mRNA vectors are safe and provide transient expression of BEs to prevent unwanted off-target effects⁸.  Here we explore the use of Akaluc^GO to visualize BE activity in vitro and in vivo following lipid nanoparticle co-delivery of gRNAs and BE-encoding mRNAs.

Methods and Results: A plasmid was cloned with a T7 promoter driving the expression of an adenine base editor (ABE), which convert adenine bases to guanine bases. Using this cloned plasmid as a template, mRNA was synthesized via in vitro transcription. HeLa cells were engineered via lentivirus to stably express the Akaluc^GO reporter and were transfected with mRNA and GO gRNA targeting Akaluc^GO or scrambled gRNA. BLI was performed 24 hours later, showing significantly higher signal in cells transfected with GO gRNA versus scrambled gRNA (Pursuasive Data File D). There was no significant difference in BLI signal comparing different mRNA to gRNA ratios (Pursuasive Data File E). Next, mice bearing two flank Akaluc^GO HeLa tumors were injected intratumorally with mRNA and GO gRNA or scrambled gRNA complexed with the reagent in-vivoJETRNA+ (Polyplus) (n=4). In this early pilot study, BLI over a 3-week period demonstrated increased signal as early as day 4 in tumors injected with mRNA and GO gRNA compared to scrambled gRNA injection (Fig. 1A), with significantly increased signal detected by day 22 (Fig. 1B).

Discussion and Conclusions: New CRISPR technologies and medicines are rapidly being developed and used clinically for the treatment of many diseases including cancer. In 2023, the first CRISPR 1.0 therapy was approved⁹, and the first CRISPR 2.0 BE clinical trial reported promising results in patients with familial hypercholesterolemia¹⁰. This field needs new tools that can assess the delivery, activity, and outcomes of next-generation CRISPR therapies both during their development and application. We have developed a GO system using the highly sensitive BLI reporter Akaluciferase to visualize base editing activity in vivo following mRNA-mediated BE delivery. Future work focuses on verifying GO reporter editing with DNA sequencing. We expect that the versatility of GO systems will be useful to evaluate new BEs and delivery vehicles/vectors as they are developed, and with further development could provide a surrogate non-invasive measure of BE treatment response.

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Image/Figure Caption:

Figure 1. Bioluminescence imaging (BLI) of Base Editing Activity using an Activatable AkalucGO Reporter. A) BLI of AkalucGO in a subcutaneous model of a HeLa kidney cancer cell line engineered with AkalucGO. The thin and thick arrows represent the tumor injected with a scrambled guide RNA (gRNA) (left) and GO gRNA targeting AkalucGO (right), respectively. B) Longitudinal BLI measurements of AkalucGO  mean radiance in both tumor regions. Data are presented as mean ± SD (“ns” non-significant, *p < 0.05) and were analyzed with Two-way ANOVA (p<0.05).

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