Abstract

Cancer is like a genetic circus gone wild, with rogue cells growing uncontrollably due to numerous mutations. Traditional cancer treatments like chemotherapy and radiotherapy are effective but also harm healthy cells, leading to severe side effects. Furthermore, as these cells accumulate mutations over time, they become increasingly resistant to those treatments. This demonstrates the urgent need of novel precise targeting tools to combat cancer.

Enters the calvary CRISPR/Cas13, a groundbreaking gene-editing tool that brings new hope. CRISPR/Cas13 targets RNA to stop cancer right at its source. CRISPR/Cas13 acts as a guided missile that is programmed to destroy abnormal mutated RNAs – they can precisely destroy cancerous RNA without affecting healthy cells. However, there are hurdles to overcome such as delivering CRISPR into the body, avoiding immune system attacks, and ensuring it doesn’t accidentally target healthy RNA.

There are multiple variants of CRISPR/Cas13 proteins initially described in bacteria, but human cells’ complex organization may affect their RNA cleavage abilities. Our research develops a smart assay using fluorescent markers and aims at tracking the precision as well as any side effects of these CRISPR variants. We report that two Cas13 variants, LwaCas13a and PspCas13b, exhibit high specificity and silences only the intended target RNA. However, a third variant, RfxCas13d, while also capable of silencing target RNA, shows very potent collateral activity against RNAs and causes cell death.

Overall, this study demonstrates the potential for some Cas13 proteins to precisely target oncogenic RNAs in human cells. This programmable RNA targeting tool holds significant promise in addressing the challenges posed by previously undruggable targets in cancer therapeutics.