Abstract
In this project, we characterized a type III-A CRISPR-Cas system present in Lactobacillus delbrueckii subsp. bulgaricus (LbCsm). Its effector complex was reconstituted by expression in Escherichia coli and purified. In vitro studies showed that the LbCsm effector cleaves the target RNA (tgRNA) at 6-nt periodicity and the single-stranded (ss) unspecific DNA in the presence of cognate tgRNA (CTR), indicating LbCsm possesses the backbone RNA cleavage activity and tgRNA-activated single-stranded deoxyribonuclease (ssDNase) activity. While LbCsm failed to yield any detectable cyclic oligoadenylate (cOA) products. In vivo studies exhibited that CTR is capable of conferring plasmid clearance to the LbCsm system albeit it only possesses the RNA-activated ssDNA cleavage activity. Mutagenesis of conserved motifs of the HD, Linker, and Palm2 domains of LbCsm1 indicated that none of these Csm1 mutations affected the effector assembly and the backbone cleavage. However, the Linker and Palm2 domains are found to involve in mediating allosteric regulation of the ternary effector to yield active enzyme. To investigate how the 3ʹ-protospacer flanking sequence (3-PFS) of CTR and non-cognate tgRNA (NTR) could regulate the LbCsm DNase, truncated derivatives of CTR (CTR-1 to CTR-6) and NTR (NTR1 through NTR6) were generated, carrying +1, +2, +3, +4, +5 and +6 nt of 3-PFS, respectively. Strikingly, both of NTR1 and CTR1 mediated a major stimulation to the LbCsm DNA binding, and they also facilitated the DNA cleavage, indicating that the first nucleotide in 3-PFS of NTR and CTR functions as a trigger to the allostery of the ternary LbCsm complexes, yielding an active effector. Thereafter, the +2 to +4 nt in 3-PFS of CTR further facilitate the substrate binding for more active DNA cleavage, +2 to +6 nt in 3-PFS of in NTR gradually deactivates the ternary effector complex.
The current knowledge of antiviral mechanisms by type III CRISPR systems indicated the cOA signalling pathway plays a main role in the immunity. We further investigated the mechanism of complete plasmid clearance by the unique type III-A system. We found that ATP functions as a ligand to enhance the ssDNA degradation by LbCsm, and the enhancement relies on the interaction between ATP and the Palm2 domain
of the LbCsm1 subunit, and the ATP-stimulated ssDNase activity is essential for the in vivo LbCsm immunity. In vivo studies further demonstrated that not only invader nucleic acid but also hosts’ nucleic acid could be targeted by the ssDNase of LbCsm. To investigate the DNA cleavage mechanism of LbCsm in vitro, a double-strand (ds) DNA containing a T7 promoter and the target sequence complementary to the crRNA as well as their flanking sequences was used as the template for transcription with T7 RNA polymerase to generate cognate target mRNA, which should guide LbCsm for DNA cleavage. We found that the cleavage sites not only at the 3ʹ-flanking side of the target region, but also on the 5ʹ-flanking side of the target or at the region with complementary to the crRNA or at the lead end of T7 promoter, indicating the activated LbCsm DNase is capable of targeting different dsDNA segments containing a transcriptional bubble independent of a target DNA sequence, in contrast to the co-transcriptional inference reported for the type III-A system of Staphylococcus epidermidis. Finally, we realized that the robust ATP-stimulated cleavage of ssDNA by LbCsm could be harnessed for efficient RNA detection and quantification. To do that, the specificity and sensitivity of this method was tested. The results showed that a single-molecule level of sensitivity was achieved in specific RNA detection, and the signal accumulation was positively correlated with the increase of target RNA concentration by the nuclease-dead Csm3 mutation complex, suggesting LbCsm exhibits an exciting opportunity to provide a potential platform for specific RNA detection and quantification
The current knowledge of antiviral mechanisms by type III CRISPR systems indicated the cOA signalling pathway plays a main role in the immunity. We further investigated the mechanism of complete plasmid clearance by the unique type III-A system. We found that ATP functions as a ligand to enhance the ssDNA degradation by LbCsm, and the enhancement relies on the interaction between ATP and the Palm2 domain
of the LbCsm1 subunit, and the ATP-stimulated ssDNase activity is essential for the in vivo LbCsm immunity. In vivo studies further demonstrated that not only invader nucleic acid but also hosts’ nucleic acid could be targeted by the ssDNase of LbCsm. To investigate the DNA cleavage mechanism of LbCsm in vitro, a double-strand (ds) DNA containing a T7 promoter and the target sequence complementary to the crRNA as well as their flanking sequences was used as the template for transcription with T7 RNA polymerase to generate cognate target mRNA, which should guide LbCsm for DNA cleavage. We found that the cleavage sites not only at the 3ʹ-flanking side of the target region, but also on the 5ʹ-flanking side of the target or at the region with complementary to the crRNA or at the lead end of T7 promoter, indicating the activated LbCsm DNase is capable of targeting different dsDNA segments containing a transcriptional bubble independent of a target DNA sequence, in contrast to the co-transcriptional inference reported for the type III-A system of Staphylococcus epidermidis. Finally, we realized that the robust ATP-stimulated cleavage of ssDNA by LbCsm could be harnessed for efficient RNA detection and quantification. To do that, the specificity and sensitivity of this method was tested. The results showed that a single-molecule level of sensitivity was achieved in specific RNA detection, and the signal accumulation was positively correlated with the increase of target RNA concentration by the nuclease-dead Csm3 mutation complex, suggesting LbCsm exhibits an exciting opportunity to provide a potential platform for specific RNA detection and quantification
| Originalsprog | Engelsk |
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| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
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| Status | Udgivet - 2019 |