Rapid PCR-based screening system for detection of type II CRISPR-Cas loci in bacterial species
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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease gene (Cas), originating from the bacteria acquired immune system, have revolutionized gene editing technology. In this regard, type II (Cas9) been extensively studied and widely applied CRISPR system so far. The mechanism for precise manipulation of genomic sequences is guided by small RNA called CRISPR RNA (crRNA). In this study we devised and optimized CRISPR-Cas9 screening system based on Cas9 gene detection, targeting a conserved part of recognition domain (REC) consisting of arginine rich bridge helix (BH). We used hemi-nested PCR approach for screening sensitivity and reproducibility. The recombinant E. coli DH5 alpha containing the pRGEB32 vector (DH5 alpha/pRGEB32) with the Cas9 gene was used for system optimization. Subsequently, the screening system was applied and validated on different environmental bacterial strains including Alcaligenes faecalis and Pseudomonas stutzeri , isolated from sewerage samples. The optimized hemi-nested PCR resulted in amplification of targeted region in environmental bacterial strains and results were reproduced successfully. Furthermore, nucleotides and amino acid sequence, motif and domain analysis of PCR products, confirmed the targeted Cas9 REC-BH domain. Presently, no rapid and cost effective CRISPR-Cas screening system is available except expensive whole genome sequencing approach. Our investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species. In this context, the developed Cas9 gene-based CRISPR-Cas screening system (C9CSS) may be a potential rapid screening tool to identify new Cas9 orthologs in different bacterial genomes with improved functions.
IMPORTANCE
The bacterial acquired immune system comprising CRISPR-Cas locus, has revolutionized targeted gene editing and now applications beyond it in the form clinical remedy including diagnostics and therapeutics for bacterial, viral and genetic diseases. Notably, Cas9 (Type II CRISPR) is the first one studied in detailed and programmed for different applications and discovery of its new orthologs with improved functions is still area of interest. But the limitation of large-scale screening of bacterial species for new orthologs identification due to expensive WGS workflow is a big challenge. To address this problem, we have developed and optimized a cost effective screening tool C9CSS to preliminary identify CIRISPR type-II locus in bacteria. Subsequently, only C9CSS positive sample may be processed for WGS for detailed characterization evading the cost of negative samples.
