CRISPR/Cas systems in cyanobacteria, their involvement in cell differentiation and potential for metabolic manipulation
Final Report Abstract
Native Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins are well characterized for their function as RNA-based adaptive and inheritable immune systems found in many bacteria and archaea. CRISPR-Cas systems are extremely diverse. Based on bioinformatic analyses, they are currently classified into 2 classes, 6 types and 33 subtypes. Multiple genetic approaches have been derived from these native CRISPR-Cas systems for the manipulation of gene expression and genome editing. Some of these systems – such as the CRISPR Cas9 and Cas12a “gene scissors” – represent the most efficient and reliable genome editing tools developed to date. However, different gene and genome editing strategies require different such tools. Therefore, there is substantial interest from the applied side as well as from the side of fundamental research to investigate novel or previously less characterized CRISPR- Cas systems. In this project, CRISPR-Cas systems native to multicellular cyanobacteria were addressed. First, we characterized these cyanobacteria as a prolific resource for novel and previously understudied CRISPR-Cas systems. During the course of the project we addressed the interactions between different systems cooccurring in one and the same organism and found evidence for molecular cross-talk between them. In the model cyanobacterium Anabaena (Nostoc) sp. PCC 7120 we found eleven CRISPR-like repeat-spacer arrays. All of them are transcribed and, based on structural analyses and the specifities of the cognate Cas6 maturation endonucleases, we could assign five of these arrays to belong to a type III-D CRISPR-Cas system and another five to a type I-D CRISPR-Cas system. Molecular cross-talk between these two different systems involves a previously unrecognized post-transcriptional mechanism. Intriguingly, we found the remaining array to belong to an entirely different CRISPR- Cas system, showing hallmarks of a CRISPR-associated transposon (CAST) system. Focusing on this system in Anabaena sp. PCC 7120, we characterized the previously undescribed protein Alr3614 in molecular detail and found that it functions as a transcriptional repressor of the CAST system. We suggest to name this family of repressors CvkR, for Cas V-K repressors. Further work suggested that CvkR is a representative of a separate subfamily of MerR-type transcriptional regulators and that such regulators are at the core of a widely conserved regulatory mechanism that controls type V-K CAST systems. The results bear potential for the development of new applications and present fundamental new insight.
Publications
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(2019) CRISPR-Cas systems in multicellular cyanobacteria. RNA Biology 16, 518-529
Hou S., Brenes-Álvarez M., Reimann V., Alkhnbashi O.S., Backofen R., Muro- Pastor A.M., Hess W.R.
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(2020) Approaches to study CRISPR RNA biogenesis. Methods (Elsevier B.V.) 172, 12-26
Behler J., Hess W.R.
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(2020) Specificities of and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups. RNA Biology 17, 1442-1453
Reimann V., Ziemann M., Li H., Zhu T., Behler J., Lu X., Hess W.R.
