Abstract CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from Streptococcus pyogenes (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the S. pyogenes CRISPR-Cas locus is cytotoxic in Lacticaseibacillus paracasei, even in the absence of SpyCas9. Deleting a putative transcription regulator in L. paracasei alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.