Identification And Functional Analysis Of CRISPR Associated Proteins

Clustered regularly interspaced short palindromic repeat(CRISPR)-CRISPR-associated proteins (Cas) are adaptive immune systems of archaea and bacteria. These systems have recently attracted much attention due to the applications of CRISPR-Cas for genome editing. The CRISPR-Cas system retains"memories" from past infections and provides specific resistance to these infections via an RNA-guided process. These immune systems show extreme diversity of Cas protein composition as well as genomic loci architecture. The most crucial step in depiction of cas operons composition is the identification of cas genes or Cas proteins. Furthermore,given the continuous increase of the newly sequenced archaeal and bacterial genomes,the recognition of new Cas proteins is becoming possible, which not only provides candidates for novel genome editing tools but also helps to understand the prokaryotic immune system better. Here, we describe the HMMCAS, a web service for the detection of CRISPR-associated structural and functional domains in protein sequences.HMMCAS uses hmmscan similarity search algorithm in HMMER3.1 to provide a fast,interactive service, and searches a comprehensive collection of Cas protein family hidden Markov models. This web service can accurately identify the Cas proteins including those fusion proteins (for example, the Casl-Cas4 fusion protein in Candidatus Chloracidobacterium thermophilum B) in the genomes of bacteria and archaea and even giant viruses. HMMCAS can also find putative cas operon and determine which type it belongs to. HMMCAS is freely available at http://i.uestc.edu.cn/hmmcas.Next, we identified the cas genes in the reference genome of bacteria and archaea via similarity search, and found the potential cas operons composed of cas genes. We classify the potential cas operons according to the unique signature gene of CRISPR-Cas system. We found that type ? system is the most widely distributed and abundant CRISPR-Cas system compared with type ?-type V systems. We also focused on the cas locus of the type ? system. According to the architecture of the cas locus, the subtypes ?-A1, ?-B1, ?-C1, ?-C2, ?-D were added. According to the components of cas locus, we deemed ?-A1 as degraded II-A system which lacked genes coding Cas2 protein compared with ?-A, while ?-B1 seemed to be degraded ?-B system lacking genes encoding Cas1?Cas2 those two proteins, and we also considered ?-C1 and ?-C2 as degraded systems without genes encoding proteins Cas2 and Casl. The phylogenetic tree of the Cas9 protein also accounts for such evolutionary relationships. In particular,although Cas9 proteins encoded by sub-type ?-D did not show single division, they all are converged together with Cas9 proteins of sub-type ?-C??-C1??-C2, which hinted that sub-type ?-D might be degraded ?-C systems or degraded ?-C1??-C2 systems.Furthermore, we also observed that ? type systems fuse with ??????? types rspectively.Finally, we discovered that cas locus contained genes encoding toxin protein,anti-toxin protein and Ago protein via annotating genes whose function remained undisclosed on cas locus. Specificly, we discovered 4 toxin protein families: AAA 21 ?AbiEii?HicA and Fic,and 4 anti-toxin protein families: PhdYeFM?MazE?Unstab and BrnA. In most of the cases we found, the antitoxin genes are located upstream of their cognate toxin genes—such an organization appears to promote production of the antitoxins at higher levels than that of their cognate toxins. For all four antitoxin families, we found cas loci containing both antitoxin and toxin genes, providing additional evidence for the immunity-dormancy/suicide coupling hypothesis. We also discovered that such correlation not only exists in type ? CRISPR-Cas system ,but also are seen in type ? stems. Furthermore, we observed the phenomenon that cas locus can encode Ago protein. These cas loci belong to type ? CRISPR-Cas system.