NEAT1-triggered collateral activity of CRISPR/Cas13 exhibits antileukemic activity Free

Numerous RNAs are highly expressed in specific types of cancers, yet many lack functional significance. Similarly, most mutations occurring in cancer cells do not contribute to tumorigenesis. These “marker RNAs” and “passenger mutations” have largely been overlooked in cancer research. Here, we propose that such non-driver RNAs and genetic alterations can serve as therapeutic targets by leveraging the collateral activity of CRISPR/Cas13.

CRISPR/Cas13 is a CRISPR-associated protein that specifically targets RNA. A unique feature of CRISPR/Cas13 is its collateral activity, wherein recognition of target RNAs induces conformational changes that activate its nuclease function, leading to cleavage of both target RNAs and bystander RNAs. This indiscriminate transcriptome destruction, termed collateral activity, has previously been utilized for nucleic acid detection in diagnostics. Although initially believed to occur only in cell-free in vitro assays, recent studies have shown that CRISPR/Cas13 can induce collateral transcriptome destruction in mammalian cells, particularly when targeting highly expressed RNAs.

In this study, we demonstrate that CRISPR/RfxCas13d, a specific subtype of Cas13, targeting the long non-coding RNA NEAT1, exhibits potent antileukemic effects through its collateral activity. NEAT1, which plays a critical role in the organization and function of nuclear paraspeckles, is highly expressed in various tumors, including leukemia. By employing CRISPR/RfxCas13d and guide RNAs (crRNAs) designed to target NEAT1, we observed significant growth inhibition across multiple myeloid and lymphoid leukemia cell lines (K562, MOLM13, THP1, U937, NB4, KASUMI1, TF-1, OCI-AML3, TS9;22, NALM6, RAJI, and SLVL) in vitro. This antileukemic effect was further validated in vivo through xenograft assays using K562 and MOLM13 cells, as well as patient-derived xenograft (PDX) cells from acute myeloid leukemia (AML) patients with high NEAT1 expression.

Interestingly, this antileukemic effect was exclusive to CRISPR/RfxCas13d targeting NEAT1. Neither NEAT1 knockdown via CRISPR interference (CRISPRi) nor NEAT1 knockout using CRISPR/Cas9 produced inhibitory effects on leukemic proliferation. Additionally, high-fidelity CRISPR/Cas13 (hfCas13), which lacks collateral activity, failed to suppress K562 cell growth. RNA-seq and RIP-seq analyses revealed extensive transcriptomic changes triggered by NEAT1-targeting CRISPR/RfxCas13d, with preferential downregulation of NEAT1-interacting RNAs. These findings suggest that NEAT1 itself is dispensable for leukemic growth, and that the collateral activity of CRISPR/RfxCas13d induces cleavage of bystander RNAs associated with NEAT1, leading to proliferation defects in leukemia cells.

We further investigated the requirements for Cas13's collateral activity in leukemia cells. NEAT1 knockdown via CRISPRi reduced NEAT1 expression to approximately 50% of parental levels in K562 cells, abolishing the antileukemic effect of NEAT1-targeting RfxCas13d. This indicates that high NEAT1 expression is necessary to trigger CRISPR/RfxCas13d's collateral activity. Additionally, introducing mutations in NEAT1 target sequences using CRISPR/Cas9 rendered K562 cells resistant to wild-type NEAT1-targeting RfxCas13d, while making them sensitive to crRNAs targeting the mutated NEAT1. Cell line-specific NEAT1 mutations were then targeted, resulting in selective growth inhibition of THP1 and U937 cells harboring the mutations with minimal impact on other cell lines. These results highlight the ability of CRISPR/RfxCas13d to selectively target leukemia-specific marker RNAs and mutations, irrespective of their functional importance.

Finally, we investigated whether other non-essential RNAs could also serve as therapeutic targets for CRISPR/Cas13. RNASE2 and MPO were identified as being highly expressed in THP1 and NB4 cells, respectively, despite neither gene being essential for leukemic proliferation. CRISPR/RfxCas13d targeting RNASE2 or MPO selectively suppressed the growth of THP1 and NB4 cells, respectively, without affecting the proliferation of other leukemia cell lines.

Collectively, our findings provide proof of concept that marker RNAs and passenger mutations can be leveraged as therapeutic targets using CRISPR/RfxCas13d. This approach substantially broadens the range of treatment options for leukemia patients and underscores the potential of Cas13's collateral activity in advancing personalized cancer therapeutics.