Cas9-independent tracrRNA cytotoxicity in Lacticaseibacillus paracasei

This study shows that the expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the Streptococcus pyogenes CRISPR-Cas locus is cytotoxic to Lacticaseibacillus paracasei, even in the absence of SpyCas9. The potential involvement of tracr-S in interacting with essential host RNAs as the cause of cytotoxicity was further investigated using MS2-affinity purification coupled with RNA sequencing (MAPS), which identified several co-purified and enriched cellular RNAs. These interactions may result in silencing effects that compromise host cell viability. This study provides new insights into the potential roles of other CRISPR-Cas9 components as sources of cytotoxicity during the heterologous implementation of SpyCas9.