| Tuberculosis is a chronic infectious disease caused by M.tb.In recent years,drugresistant tuberculosis has occurred frequently due to the abuse of antibiotics,seriously threatening public health and social safety.The new treatment methods are more urgent because existing anti-tuberculosis drugs can no longer meet the need for the treatment of drug-resistant tuberculosis.In recent years,the CRISPR-Cas system has become a research hotspot due to its huge application potential in the field of gene editing.M.tb has a type Ⅲ-A CRISPR-Cas system,and its endogenous CRISPR-Cas system is significant to the genetic operation system and the prevention and treatment of tuberculosis.The research and application of M.tb bacteriophage began as early as 1938.M.tb bacteriophage was used for the detection of tuberculosis due to its specific infectivity,and it was used to deliver knockout elements because of its efficient infection.It has also been reported that M.tb bacteriophage was used to treat tuberculosis and achieved certain therapeutic effects.But so far there has been no report that the combination of M.tb type Ⅲ CRISPR-Cas system and Mycobacterial bacteriophage for the prevention and treatment of tuberculosis.This study sets out to use the Ⅲ-A type CRISPR-Cas system of M.tb by design a mini-CRISPR with a spacer target M.tb genome,using phage as a vector to deliver mini-CRISPR to activate the type Ⅲ-A CRISPR-Cas system interference function to achieve the killing of M.tb.At the same time,we also conducted a preliminary study on the acquisition function of the type Ⅲ-A CRISPR-Cas system of M.tb.In this study,the pMV261 plasmid with mini-CRISPR and the phAE159 bacteriophage were constructed,and the interference efficiency of mini-CRISPR was respectively detected by electrotransformation and infection.It was found that mini-CRISPR does not affect the transformation efficiency of M.tb,but mini-CRISPR can significantly reduce the mRNA level of the target gene,which shows that M.tb CRISPR-Cas system has a certain interference function and can cut RNA,but Its DNA cutting ability is limited.In addition,we overexpressed Cas1Cas2 protein in M.bovis to study the acquisition function of CRISPR-Cas system.However,PCR testing and high-throughput testing have not found any new spacers.It is worth mentioning that in the process of constructing phage vectors,we found that a new type of vector construction technologyTEDA method-was used for the construction of phage vectors.Compared with the traditional method that packaging in vitro,this method has simple and quick steps,efficient features,and very low construction costs.In this study,we also study on the bacteriophage of M.tb.We found that kanamycin inhibited the infection efficiency of phage D29 when we studied the gp69 gene of phage D29.Further research found that kanamycin,hygromycin and streptomycin can inhibit the infection efficiency of bacteriophage D29 and phAE159,and the inhibitory ability increases with the increase of antibiotic concentration.This inhibition is detected in M.smegmatis,M.tb and M.bovis.Comparing with E.coli phages shows that the aminoglycoside is specific for inhibiting the infection of mycobacterial phages.We localized this inhibition phenomenon at the stage of phage DNA injection into the host to DNA replication by using absolute quantitative PCR to detect phage DNA.However,due to the lack of technical,we have not yet accurately located the exact position of action and the molecular mechanism of its inhibition.This result suggests that when studying the bacteriophage of M.tb in the treatment of tuberculosis,the effect of antibiotics on the bacteriophage needs to be considered.Similarly,when using bacteriophage to detect drug-resistant M.tb infection,It is slao should be attention to the effect of antibiotics on bacteriophage.At the same time,this result also reminds us that antibiotics not only affect bacteria,but also affect bacteriophages. |
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