Abstract
RNA therapies using RNA editing and interference are currently being developed for neurological diseases. The CRISPR-Cas13 system, based on bacterial enzymes, holds great promise for developing efficient tools for RNA therapies. However, neurotoxic activity has been reported for Cas13a, and recent studies have reported toxic effects of PspCas13b and RfxCas13d during zebrafish and Drosophila embryonic development. It is important to investigate the safety of these bacterial enzymes in the context of the nervous system and neuronal development. In this study, we used mouse cerebellar Purkinje cells as a complex neuron type to test for the potential neurotoxic actions of RfxCas13d and PspCas13b. We found that PspCas13b significantly impeded the dendritic development of cultured Purkinje cells, similar to the neurotoxic action of Cas13a. In contrast, RfxCas13d did not exhibit a significant inhibition of dendritic development. A similar trend was found for axonal outgrowth. These results suggest varying neurotoxic properties for different Cas13 ortholog enzymes. We call for more studies to investigate, and possibly mitigate, the neurotoxicity of Cas13 proteins in order to improve the safety of the CRISPR-Cas13 system for RNA therapies.
Highlights
Our results demonstrate that RfxCas13d expression did not cause a significant reduction in Purkinje cell dendritic development, but that PspCas13b protein had a marked neurotoxic effect on the development of cultured Purkinje neurons with a significant inhibition of dendritic growth
Our findings show that RfxCas13d has no or only small neurotoxic effects on developing dendrites of cerebellar Purkinje cells, in contrast to PspCas13b or LwaCas13a [13]
We evaluated the possible neurotoxic actions of two Cas13 endonucleases by expressing the CRISPR-RfxCas13d and PspCas13b proteins in Purkinje cells and analyzing their dendritic development
Summary
RNA editing technologies offer a novel way to manipulate RNA information without altering DNA [1,2,3,4]. While most of the initial studies on gene-knockdown using Cas were done with Cas13a, more recently Cas13d and Cas13b were identified as related Cas enzymes of the Cas protein family in bacteria. These enzymes contain two higher eukaryote and prokaryote nucleotide-binding domains associated with ribonucleases [5,6,7,8,9,10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
