Detection Of Known CRISPR-Cas System And Discovery Of Unknown Cas Protein

The CRISPR-Cas system is an RNA-guided sequence-specific endonuclease system,which provides adaptive immune defenses during the evolution of prokaryotes and targets and cleaves invading mobile genetic elements such as phages.Since the discovery of CRISPR-Cas systems,numerous research work has been performed and the CRISPR system has been successfully developed as new tools for genome editing,nucleic acid detection and beyond.Different from other genome editing systems such as the Zinc Finger Nuclease(ZFN)and the Transcription Activator-Like Effector Nuclease(TALEN),the CRISPR technology has been widely used in various fields because of its advantages in programmability,low cost and easy operation.Although the CRISPR-Cas9,CRISPR-Cas12 and CRISPR-Cas13 are well characterized and the associated editing systems are well established,little is known about their origin and evolution.Besides,as most bacteria/archaea cannot be cultured in the laboratory,there are probably many different types of CRISPR-Cas systems in these uncultured organisms that have not been discovered.In this thesis,I provided an overview of the development,composition,classification and functions of the CRISPR-Cas systems,and also developed a custom computational pipeline which based on HMMER alignment and Cas protein map for both type assignment of known CRISPR-Cas gene clusters and discovery of unknown Cas proteins.Researchers can directly input genomic/metagenomics assembly data(Scaffolds/Contigs),and after the calculation pipeline detects CRISPR locus and Cas homologous protein comparison steps,they can finally obtain the known CRISPR-Cas system related data contained in the sequence and Information about the unknown Cas protein.In addition,this article has conducted a detailed analysis of the two sets of data sets from different sources through the calculation pipeline,and verified the detection ability of the calculation pipeline on the known CRISPR-Cas system,nine proteins(>800aa)that may have CRISPR-Cas related functions were obtained,and the CRISPR gene clusters where the most likely three Cas proteins were located were proposed to have possible CRISPR immune mechanism conjectures.