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Published on: December 19, 2010
Cas9-independent tracrRNA cytotoxicity in Lacticaseibacillus paracasei
Adini Q Arifah1, Justin M Vento2, Isabella Kurrer1
1Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Center for Infection Research, 97080 Würzburg, Germany.
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.
Insights
The short trans-activating CRISPR RNA (tracr-S) from Streptococcus pyogenes is cytotoxic in Lacticaseibacillus paracasei, even without the Cas9 enzyme. This cytotoxicity is linked to host RNA interactions and can be alleviated by genetic modification.
Area of Science:
- Bacteriology
- Molecular Biology
- Genetics
Background:
- CRISPR-Cas9 systems are vital for bacterial genome editing but can cause cytotoxicity when expressed heterologously.
- The Cas9 nuclease (SpyCas9) is a common source of this cytotoxicity, but effects of other CRISPR components are unknown.
Purpose of the Study:
- To investigate the cytotoxic effects of other CRISPR-Cas9 system components beyond the Cas9 nuclease.
- To determine if the trans-activating CRISPR RNA (tracr-S) from Streptococcus pyogenes induces cytotoxicity in Lacticaseibacillus paracasei.
Main Methods:
- Investigated the cytotoxicity of tracr-S expression in Lacticaseibacillus paracasei.
- Analyzed the effects of deleting a putative transcription regulator on tracr-S cytotoxicity and tracr-L expression.
- Examined the specificity of tracr-S cytotoxicity and its interaction with host RNAs.
Main Results:
- Expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from S. pyogenes is cytotoxic in L. paracasei, independent of SpyCas9.
- Deleting a specific transcription regulator in L. paracasei reduced tracr-S cytotoxicity and induced the long isoform (tracr-L).
- tracr-S cytotoxicity was sequence-specific and involved direct interactions with host RNAs.
Conclusions:
- Components of CRISPR-Cas systems beyond Cas9, such as tracr-S, can cause cytotoxicity in heterologous bacterial hosts.
- These findings have implications for the application of heterologous CRISPR-Cas systems in bacteria and the evolution of CRISPR immunity.
