CRISPR-Cas13d-Mediated Targeting of a Context-Specific Essential Gene Enables Selective Elimination of Uveal Melanoma

Abstract

Uveal melanoma, the most common eye cancer in adults, remains limited to surgical intervention and chemotherapy, with a dismal survival rate that has not improved in over 50 years. To address this therapeutic impasse, we systematically analyzed public gene expression, RNAi, and CRISPR knockout datasets and identified RASGRP3 as an essential gene specifically for uveal melanoma. RasGRP3 is uniquely overexpressed and essential for survival in uveal melanoma cells, but dispensable in healthy cells. RasGRP3 remains "undruggable" due to its intracellular localization and lack of targetable binding pockets. To overcome this, we developed a CRISPR-Cas13d RNA-targeting therapeutic that specifically knocks down RasGRP3 mRNA. This Cas13d-based therapeutic mediates selective uveal melanoma killing through two synergistic mechanisms: (i) direct silencing of the essential RasGRP3 transcript, and (ii) collateral RNA degradation triggered by the cleavage of overexpressed RasGRP3. When delivered via optimized lipid nanoparticles encoding Cas13d mRNA and guide RNA, this strategy eliminated >97% of uveal melanoma cells while sparing healthy cells, including retinal pigment epithelial cells. This approach outperformed conventional Cas9 and siRNA methods in potency without inducing permanent genomic alterations. Our findings establish a RNA-targeting therapeutic for uveal melanoma and a framework for Cas13d-based interventions against broad "undruggable" cancers.

Keywords: CRISPR-Cas13d; RNA targeting; cancer therapeutic; context-specific essential gene; precision tumor targeting; uveal melanoma.

Figure 1:

Bioinformatic screening reveals RasGRP3 as a highly-expressed context-specific essential gene for uveal melanoma. (A) Diagram of target identification pipeline using CRISPR-Cas9 cancer dependency screens. (B) Uveal melanoma proliferation pathway and the function of RasGRP3. (C) CRISPR-Cas9 knockout screen data reveals that uveal melanoma is highly dependent on RasGRP3. (D) RNA-seq data shows that eye cancer (from TCGA) exhibits several-fold higher expression of RasGRP3 than both other cancers and healthy tissues (GTEx).

Figure 2:

Targeting RasGRP3 using CRISPR-Cas13d yields potent and selective killing of uveal melanoma cells. (A) Diagram of Cas13’s mechanism of action: once the gRNA binds to the target mRNA, it will degrade the target mRNA and surrounding off-target mRNA. (B) Schematic of methods used in (C) and (D). Cas13d is lentivirally transduced into cells, followed by transfection of individual guides to induce knockdown of RasGRP3. Cell viability is quantified 48 hours after transfection. (C) Viability assay in 92-1 (UM) cells to screen the efficacy of 42 different gRNAs targeting RasGRP3, along with three non-targeting gRNAs. (D) Viability assay in both 92-1 (UM) and HEK 293T (non-UM) cells to examine gRNA specificity using the eight best gRNAs from (C), along with a non-targeting gRNA and untransfected control. Each data point represents one biological replicate. (E) RasGRP3 gene map with each targeting gRNA screened in (C) annotated based on the location it targets.

Figure 3:

Cas13d mRNA and RasGRP3-targeting gRNA delivered via a therapeutically relevant LNP induce UM-specific cell death. (A) Schematic of methods for (B). GFP mRNA is packaged into each LNP and transfected into uveal melanoma cells to examine delivery efficiency via fluorescence. (B) Transfection efficiency of 30 different LNP formulations in two uveal melanoma cell lines (MP-46, 92-1). The arrow is pointing to Formulation #7 (3060i10), which is used in subsequent experiments. Each data point represents one biological replicate. (C) Schematic of methods for (D). The therapeutic is formed by encapsulating Cas13d mRNA and RasGRP3-targeting gRNA in an LNP. The LNP is transfected into three uveal melanoma cell lines and three non-uveal melanoma cell lines, and then cell death is measured via flow cytometry after 48 hours. (D) In-vitro viability assay in three uveal melanoma cell lines (92-1, MP-41, MP-46) and three non-UM cell lines (HEK-293T, HeLa, ARPE-19). Each data point represents one biological replicate.

Figure 4:

On-target mRNA knockdown and collateral cleavage by Cas13 contribute synergistically to cytotoxicity in cancer cells. (A) Schematic of methods for (B). Cas13d or SpCas9 are lentivirally integrated into 92-1 (UM) cells, followed by transfection of their corresponding guides. Alternatively, siRNA is transfected into the cells. Cell viability is quantified 48 hours after transfection by performing a Live/Dead stain followed by flow cytometry. (B) Comparison of Cas9 knockout, siRNA knockdown and Cas13d knockdown of RasGRP3 in 92-1 (UM) cells. Two RasGRP3-targeting guides and one non-targeting guide were tested for each CRISPR enzyme, along with RasGRP3-targeting siRNA and non-targeting siRNA. (C, D) qPCR results 12 hours after transfection of the Cas13d-LNP therapeutic into ARPE-19 (non-UM) and three UM cells (MP-46, MP-41, 92-1) (N=4). Expression relative to MTATP6 (mitochondrial RNA) is shown for (C) RasGRP3, and (D) GAPDH (off-target RNA for collateral cleavage). (E) Schematic of methods for (F). A sequence-modified, guide-orthogonal RasGRP3 gene (i.e., not targetable by targeting guide, but the same protein is produced) is stably integrated into uveal melanoma cells (92-1). The Cas13d-LNP therapeutic is then transfected, leading to degradation of the original RasGRP3 mRNA, but not the modified version. Viability of the cells is then quantified 48 hours after transfection to compare the impacts of collateral cleavage and RasGRP3 dependency. (F) In-vitro viability assay in modified uveal melanoma cells to determine the mechanism of cell killing by Cas13d (RasGRP3 knockdown vs. collateral cleavage).

Figure 5:

A pipeline for developing Cas13 therapeutics against cancer. A search through CRISPR-Cas9 KO, RNAi, and other cancer dependency screens yields initial targets, which can be filtered to find context-specific and undrugged fitness genes. Once the target has been identified, perform a gRNA screen to find the most effective/selective targeting guide. Finally, encapsulate the Cas13 mRNA and targeting gRNA into an LNP (or other delivery vehicle), creating a new Cas13 cancer therapeutic.