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Updated: Nov 12, 2025
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
crRNA complementarity shifts endogenous CRISPR-Cas systems between transcriptional repression and DNA defense
Hannah K Ratner1,2,3, David S Weiss2,3,4
1Microbiology and Molecular Genetics Program, Emory University, Atlanta, GA, USA.
Abstract:
CRISPR-Cas systems are prokaryotic adaptive immune systems that recognize and cleave nucleic acid targets using small RNAs called CRISPR RNAs (crRNAs) to guide Cas protein(s). There is increasing evidence for the broader endogenous roles of these systems. The CRISPR-Cas9 system of Francisella novicida also represses endogenous transcription using a non-canonical small RNA (scaRNA). We examined whether the crRNAs of the native F. novicida CRISPR-Cas systems, Cas12a and Cas9, can guide transcriptional repression. Both systems repressed mRNA transcript levels when crRNA-target complementarity was limited, and led to target cleavage with extended complementarity. Using these parameters we engineered the CRISPR array of Cas12a to guide the transcriptional repression of a new and endogenous target. Since the majority of crRNA targets remain unidentified, this work suggests that a re-analysis of crRNAs for endogenous targets with limited complementarity could reveal new, diverse regulatory roles for CRISPR-Cas systems in prokaryotic biology.
Insights
CRISPR-Cas systems can regulate gene expression by guiding transcriptional repression. This study shows CRISPR RNAs can direct Cas proteins to repress target genes, revealing new roles for these systems in prokaryotes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- CRISPR-Cas systems are prokaryotic adaptive immune systems utilizing CRISPR RNAs (crRNAs) and Cas proteins for nucleic acid targeting.
- Endogenous roles of CRISPR-Cas systems beyond immunity are increasingly recognized.
- The CRISPR-Cas9 system in *Francisella novicida* utilizes a small RNA (scaRNA) for transcriptional repression.
Purpose of the Study:
- To investigate if native CRISPR-Cas systems (Cas12a and Cas9) in *F. novicida* can guide transcriptional repression using their crRNAs.
- To explore the relationship between crRNA-target complementarity and the outcome (repression vs. cleavage).
- To engineer a CRISPR-Cas12a system for targeted endogenous gene repression.
Main Methods:
- Analysis of *F. novicida* Cas12a and Cas9 systems' ability to guide transcriptional repression.
- Varying crRNA-target complementarity to observe effects on gene expression.
- Engineering the CRISPR array to direct Cas12a for repression of a specific endogenous target.
Main Results:
- Both Cas12a and Cas9 systems repressed mRNA transcript levels when crRNA-target complementarity was limited.
- Target cleavage occurred with extended complementarity between crRNA and target.
- Engineered Cas12a system successfully repressed a new endogenous target.
Conclusions:
- CRISPR-Cas crRNAs can guide transcriptional repression, not just cleavage, depending on complementarity.
- This suggests a broader regulatory function for CRISPR-Cas systems in prokaryotes.
- Re-analysis of crRNAs may uncover novel regulatory roles and endogenous targets, especially those with limited complementarity.
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