Technical Review: Engineering circular guide RNA and CRISPR-Cas13d-encoding mRNA for the RNA editing of Adar1 in triple-negative breast cancer immunotherapy

Authors: Shurong Zhou, Suling Yang, Jie Xu, Guizhi Zhu

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, USA

Background Introduction

This research falls under the broader scientific discipline of molecular biology and cancer therapeutics, specifically within RNA-guided genome editing and precision oncology. CRISPR-Cas13d systems represent a powerful class of RNA-targeting tools that can precisely edit target transcripts with minimal off-target effects. However, their clinical development faces significant challenges. One of these is the limited biostability of conventional linear guide RNAs (lgRNAs) susceptible to enzymatic degradation, the immunogenicity of prokaryotic Cas proteins, and safety concerns from prolonged Cas expression. The research specifically targets ADAR1 (Adenosine Deaminase Acting on RNA type I), a critical RNA editor that converts adenosine to inosine in double-stranded RNA, which plays essential roles in immune homeostasis and has been identified as overexpressed in various cancers such as triple-negative breast cancer (TNBC).

Relative to lgRNA, cgRNA showed enhanced biostability and improved Adar1 knockdown efficiency in cells. A) Left: predicted secondary structure of three different engineered gRNA; right: Adar1 RNA levels of 4T1 cells 24 h after transfection with mRNA-RfxCas13d (1 μg/mL) with different gRNA (1 μg/mL). RNA was transfected using Lipofectamine 2000. B) PAGE gel electrophoresis of lgRNA and cgRNA before and after RNase R treatment (15 min). C) RNA integrity analysis of lgRNA and cgRNA based on agarose gel of RNA treated with diluted FBS. Bottom: a representative agarose gel image; top: summarized fractions of intact RNA based on three independent replicates of agarose gel results. D) Adar1 RNA levels in 4T1 cells 24 h after treatment with mRNA-RfxCas13d (0.5 μg/mL) and lgRNA or cgRNA (0.25 μg/mL) at low concentrations. E) Adar1 RNA levels in 4T1 cells 24 h after treatment with mRNA-RfxCas13d with lgRNA, cgRNA or 2’-OMe modified gRNA. Data: mean ± SD. n.s.: not significant, p> 0.05; p < 0.05, *p < 0.01; ***p < 0.001. p value was determined by an unpaired student t-test.

Materials and Methodology

The researchers optimized cgRNA sequences for CRISPR-Cas13d editing through knockdown efficiency testing in 4T1 murine TNBC cells. The cgRNA in question was synthesized through enzymatic ligation using T4 RNA ligase I, converting linear gRNA precursors into stable circular structures. After optimization, the team used lipid nanoparticles to deliver the mRNA-RfxCas13d. This was first done to various cell lines (4T1 murine TNBC cells, MC38 murine colorectal cells, and MDA-MB 231 human TNBC cells). Subsequently, a 4T1 murine TNBC tumor model was used for in vivo studies, with mRNA-LNPs regularly administered and tumor volumes continuously monitored.

Results

Circular Guide RNA Characterization

The cgRNAs demonstrated significantly enhanced biostability compared to linear counterparts, with over 90% intact cgRNA remaining after 15 minutes in PBS containing fetal bovine serum, versus approximately 50% for lgRNA. Despite the circular structure, cgRNAs maintained comparable Cas13d-binding affinity to lgRNAs in EMSA assays and remained structurally intact when complexed with RfxCas13d protein, avoiding early linearization.

In Vitro ADAR1 Targeting Efficacy

At lower gRNA concentrations (0.25 µg/mL), cgRNA achieved approximately 45% ADAR1 knockdown efficiency, representing a 2.2-fold improvement over lgRNA. cgRNAs outperformed both lgRNAs and 2'-O-methyl modified gRNAs in knockdown efficiency. ADAR1 protein reduction reached as low as 37% around 3 days post-transfection, returning to baseline by 5 days, demonstrating transient expression. Importantly, no collateral RNA cleavage activity was detected in dual reporter cell assays, confirming the specificity of the system.

Immunosensitization Effects

Treatment with cgRNA + mRNA-RfxCas13d significantly sensitized TNBC cells to cytokine-mediated apoptosis. Following ADAR1 knockdown and subsequent IFN-γ treatment, treated 4T1 cells showed increased apoptosis and reduced cell viability compared to controls, with no reduction in cell viability observed in the absence of IFN-γ treatment.This demonstrates the therapeutic mechanism whereby ADAR1 targeting enhances cancer cell sensitivity to immune-mediated killing, crucial for combination immunotherapy approaches.

In Vivo Therapeutic Efficacy

IVIS imaging confirmed tumor-specific expression of LNP-delivered mRNA for at least 3 days, with primary uptake in CD45⁻ EpCAM⁺ cells (likely 4T1 tumor cells). In the therapeutic study, mRNA-RfxCas13d + cgRNA markedly enhanced the tumor inhibition efficacy of anti-CTLA-4 therapy, with cgRNA significantly outperforming lgRNA in combination with immune checkpoint blockade. No mouse body weight loss was observed during treatments, suggesting promising safety profiles.

Conclusion

Zhou and colleagues demonstrated that circular guide RNAs combined with mRNA-encoded CRISPR-Cas13d can overcome key limitations of conventional linear guide RNA systems. The enhanced biostability of cgRNAs, coupled with transient mRNA-based Cas13d expression, provides an improved therapeutic platform for precise RNA editing with reduced immunogenicity and off-target concerns.

This paper utilized the PreciGenome NanoGenerator Flex-S for lipid nanoparticle preparation. Precise formulation of the co-delivery system was essential for the therapeutic efficacy, and high efficiency is necessary in early stage research where extensive screening and low product volumes are required.

For further details, check out the link to the article here:

https://www.biorxiv.org/content/10.1101/2025.07.22.666181v1.abstract