Characterization And Function Analysis Of CRISPR-Cas System On Bacillus Cereus Group

Bacillus thuringiensis is a gram positive bacteria,belonged to the Bacilluscereus group.Bt is widely used in biological prevention and control as its forms a parasporal crystal during the period of spore formation.Bt pesticide often has a short duration due to the abundant phage in soil that could infect Bt strains.There are many kinds of resistance mechanisms of host to phage infection,such as abortion infection,restriction modification system and so on.CRISPR-Cas system as the immune system of prokaryotic cells,widely exists in bacteria genomes,which can prevent the invasion of exogenous phages and plasmids.Previous studies on the action mechanism of CRISPR-Cas system revealed that the process can be divided into three distinct phases:(1)the acquisition of spacers following exposure to foreign nucleic acids,(2)the transcription of spacers and transcript processing to produce a crRNA that guides target recognition,and(3)target recognition and cleavage.The current classification of CRISPR-Cas systems is based on the sequences of the cas genes,the sequences of the repeats within the CRISPR arrays,and the organization of the cas operons.The classification distinguishes three main types of CRISPR-Cas systems:Type Ⅰ,Type Ⅱ and Type Ⅲ.Type Ⅱ system only need Cas9 protein to degrade foreign DNA,thus contributing it to widely be used as gene editing system.In addition to gene editing,the CRISPR-Cas system was also being indicated the functional role in bacterial physiological process,such as the CRISPR-Cas system can enhance the virulence of Francisella.novicida,inhibit Pseudomonas aeruginosa PA14 biofilmformation,etc.1.Bc group has functional CRISPR-Cas systemIt is found that with the method of bioinformatics analysis,there are three complete Type I CRISPR-Cas systems in Bacillus.cereus group.We selected B.cereus VD115,which has a complete Type I-C CRISPR-Cas system,as material and carried on related research to explore its function:1)the RT-PCR results demonstrated that Cas protein of VD115 CRISPR-Cas system has transcripted and expressed;2)It is comfirmed that the PAM site of Type I-C CRISPR-Cas system in VD115 is TTN vias bioinformatics analysis of PAM site;3)plasmid transformation experiment confirmed the immune function of TypeICRISPR-Cas system in VD115,and this system is also functional in BMB1685,a mutant of Bacillus thuringiensis YBT-1518 harboring the Type I CRISPR-Cas system of VD115;4)co-culture BMB1685 with phages(purified phage and mixtures)showed that BMB1685 acquires resistance to phages in a very shot time;5)Agarose gel electrophoresis,T-A cloning sequencing and high-throughput sequencing illustrated that BMB1685 acquired new spacer while culturing with phages.Based on this,a functional CRISPR-Cas system can be introduced to Bt engineering strain and improve its resistance to phages.2.Gene editing use Type I-C system of B.cereus VD115It is very difficult to preform genetic operation on Bt strains,and the safety of Bt engineering bacteria has been controversial.While CRISPR-Cas system has been used as gene editing because of its high efficiency and specificity.It is demenstrated that the CRISPR-Cas system of VD115 is a functional element,so we can make full use of this system as gene editing tool.As tracrRNA is not invoved in TypeⅠ system,so the construction of the gene editing tool is more easy to operate.we successfully knockt out cry6Aa gene in Bt wild strain YBT-1518 via the TypeⅠ-C system,which demonstrated that the Type I-C system of B.cereus VD115 can be used as effective gene editing tool,at the same time,the problem of genetic operation on Bt genome can be solved.3.CRISPR-Cas system of Bc group has a negetive impact on environmental adaptationIn order to find out the potential function of CRISPR-Cas system in Bc group,We have detected the growth situation and environmental adaptability of BMB1685,strain contains Type I-C system from B.cereus VD115,and found that:1)the CRISPR-Cas system inhibit the growth of YBT-1518;2)the community characteristics such as biofilmformtion and swarmming are significantly decreased;3)a significant reduction of resistant ability in the high-salt and acid environment;4)reduced colonization in nematode.220 genomes of Bc group was analysisd,we found that only about 12%of the strains has CRISPR-Cas system,is far less than 45%of the bacterias.And most of the system in Bc group is not complete,mainly display on the deletion of Casl,Cas2 and Cas4 proteins,which means this system can not acquire new spacers.CRISPR-Cas system widely exist in 84%of archaea and 45%of bacteria genome,and pathogens account for the majority of the ratio.Archaea and pathogenic bacteria living environment is relatively single,don’t need to get moving components to adapt to the diverse environment.Therefore,in combination with the above experimental results,we believe that the CRISPR-Cas system in Bc group is not conducive to the adaptation of the host bacteria to the environment.