EcCas6e-based antisense crRNA for gene repression and RNA editing in microorganisms

Abstract

Abstract Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA–mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA–mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e–crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e–crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.