Anti-tracrRNA Prediction In Phage

In nature,the competition between hosts and parasites has always existed.The host must constantly evolve new mechanisms to fight against parasites to avoid extinction.In turn,parasites have evolved new mechanisms to counter these defensive systems.This battle for survival is reflected in the co-evolutionary dynamics of bacteria-phage,in which bacteria evolved the innate immune system and adaptive immunity(CRISPR-Cas system).The CRISPR-Cas system features sequence-specific targeting and degradation of invading genetic elements by CRISPR RNA(crRNA).At the same time,the phage encodes an anti-CRISPR protein(Acr)that is resistant to the CRISPR-Cas system to invade the bacteria.Due to the unique mechanism of function of Acr protein,it can be applied to the construction of transcriptional repression systems as well as specific inhibitors of gene editing to improve the safety of gene therapy.The Type II CRISPR-Cas9 system has widely been used in gene therapy,disease model construction,clinical diagnosis and bio-reactors due to its high efficiency,rapidity,simplicity and high specificity.The CRISPR-Cas9 system mainly consists of three core components,in which the trans-activated crRNA(tracrRNA)has a conserved function of promoting crRNA maturation and spacer integration,which plays an important role in the function of the CRISPR-Cas9 system.At present,only Acr protein has been identified against the anti-CRISPR system.The inhibitors based on nucleic acid-level have not yet been discovered.The inhibition of nucleic acid levels is a more efficient evolutionary approach than protein level inhibition and no translation is needed and more easy and quick than Acr.In view of the successful applications of RNAi in plants and eukaryotes,we intend to search for transcripts against CRISPR in phage genome.The prediction of tracrRNA of all bacterial genomes was accomplished by bioinformatics methods and the results were BLASTed with all phage genome sequences to find phages that are highly homologous to tracrRNA.PROKKA's CDS prediction,BPROM's promoter prediction and transcription and expression analysis identified a known and six unknown phages with potential anti-tracrRNA.None of the homologous sequences of the six phages are located in the gene coding region(CDS),in which three phage homologous sequence are located between the two CDS and all contain adjacent promoter sequences,indicating homologous sequences of the three phages may be located in a part of non-coding RNA to regulate the function of tracrRNA.However,BLAST analysis indicated that the alignment of the tracrRNA from two phage-derived bacteria and homologous sequences was not very high.The results predicted by IntaRNA indicate that the predicted anti-tracrRNA of the known phage RAP44 can form a stable interaction with tracrRNA,which can be used for future experimental verification.