Precision Targeting of Na-K-2Cl cotransporter (NKCC1) RNA In Vitro with RfxCas13d

Abstract

The Na-K-2Cl cotransporter (NKCC1) is considered an attractive drug target in the Central nervous system (CNS) for treating various CNS disorders. However, the specific role of NKCC1 in different types following injury within the CNS is not well understood due to its expression in multiple cell types. Additionally, there is a lack of a robust method for knocking down NKCC1 transcripts. In this study, we utilized Cas13 nucleases, a type of programmable RNA-targeting CRISPR enzyme, to effectively degrade NKCC1 mRNA in cultured cells. We developed a versatile pipeline for crRNA screening and validation in vitro and demonstrated the successful knockdown of NKCC1 using RfxCas13d. Our findings establish RfxCas13d as a powerful tool for targeting specific transcripts in vitro. By demonstrating the successful in vitro application of RfxCas13d-mediated NKCC1 RNA knockdown, we have laid the groundwork for future investigations into the therapeutic potential of NKCC1 modulation in CNS disorders.