Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.
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ABSTRACT:
SUBMITTER: Chen L
PROVIDER: S-EPMC8255852 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Highly sensitive, specific, rapid, and easy-to-use diagnostic methods for the detection of nucleic acids of pathogens are required for the diagnosis of many human, animal, and plant diseases and environmental monitoring. The approaches based on the use of the natural ability of bacterial CRISPR/Cas9 systems to recognize DNA sequences with a high specificity under isothermal conditions are an alternative to the polymerase chain reaction method, which requires expensive laboratory equipment. The development of the methods for signal registration with the formation of a DNA/RNA/Cas9 protein complex is a separate bioengineering task. In this work, a design was developed and the applicability of a biosensor system based on the binding of two dCas9 proteins with target DNA sequences (without their cutting) and detection of their colocalization using reporter systems based on split enzymes was studied. Using the methods of molecular modeling, possible mutual positions of two dCas9 proteins at a detectable locus of genomic DNA, allowing the split enzyme domains attached to them to interact in an optimal way, were determined. The optimal distances on DNA between binding sites of dCas9 proteins in different orientations were determined, and the dependence of the complex structure on the distance between the binding sites of dCas9 proteins was modeled. Using the methods of bioinformatics, the genomes of a number of viruses (including SARS-CoV-2) were analyzed, and the presence of genomic loci unique to the species, allowing the possibility of landing pairs of dCas9 proteins in optimal positions, was demonstrated. The possibility of a combined use of dCas9 proteins from different bacteria to expand the spectrum of detected loci was analyzed. The results of the work indicate a fundamental possibility of the creation of highly specific nucleic acid biosensors based on a combination of CRISPR/Cas9 technologies and split enzymes.
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Project description:Novel CRISPR-Cas systems possess substantial potential for genome editing and manipulation of gene expression. The types and numbers of CRISPR-Cas systems vary substantially between different organisms. Some filamentous cyanobacteria harbor > 40 different putative CRISPR repeat-spacer cassettes, while the number of cas gene instances is much lower. Here we addressed the types and diversity of CRISPR-Cas systems and of CRISPR-like repeat-spacer arrays in 171 publicly available genomes of multicellular cyanobacteria. The number of 1328 repeat-spacer arrays exceeded the total of 391 encoded Cas1 proteins suggesting a tendency for fragmentation or the involvement of alternative adaptation factors. The model cyanobacterium Anabaena sp. PCC 7120 contains only three cas1 genes but hosts three Class 1, possibly one Class 2 and five orphan repeat-spacer arrays, all of which exhibit crRNA-typical expression patterns suggesting active transcription, maturation and incorporation into CRISPR complexes. The CRISPR-Cas system within the element interrupting the Anabaena sp. PCC 7120 fdxN gene, as well as analogous arrangements in other strains, occupy the genetic elements that become excised during the differentiation-related programmed site-specific recombination. This fact indicates the propensity of these elements for the integration of CRISPR-cas systems and points to a previously not recognized connection. The gene all3613 resembling a possible Class 2 effector protein is linked to a short repeat-spacer array and a single tRNA gene, similar to its homologs in other cyanobacteria. The diversity and presence of numerous CRISPR-Cas systems in DNA elements that are programmed for homologous recombination make filamentous cyanobacteria a prolific resource for their study. Abbreviations: Cas: CRISPR associated sequences; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; C2c: Class 2 candidate; SDR: small dispersed repeat; TSS: transcriptional start site; UTR: untranslated region.
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