Microbial High-throughput Genome Deletion Based On CRISPR-Cas9 And Non-homologous End Joining

Genome editing is an important technology for us to understand and engineering microorganisms,and it is also the research focus area of modern biotechnology.The original gene knockout technology was mainly based on homologous double exchange,and the final knockout strain was obtained by positive and negative screening.Later,with the discovery of high-efficiency phage recombinase,efficient gene replacement can be achieved by one round homologous recombination(HR)that improves the efficiency of gene knockout,but this process still requires screening of knockout strains using selective marker genes and leaves a knockout "scar" on the genome.In recent years,the CRISPR-Cas gene editing technology has been developed to achieve efficient and seamless editing of genes,and it has been rapidly expanded to genetic modification of various microorganisms,setting off a revolutionary frenzy in the field of gene editing.The CRISPR-Cas system is an adaptive immune system widely exist in bacteria and archaea.The crRNA sequence guides Cas effector protein to target and cleave of specific DNA or RNA,thus resisting the invasion of foreign genetic component.Through design of crRNA sequence,we can direct the Cas protein cleave of specific DNA sequence on the genome and form a DNA double-strand break(DSB),finally editing genome during the repair of DSB.Therefore,efficient gene editing technique based on CRISPR-Cas system was developed.In eukaryotes,DSB can be repaired by both non-homologous end joining(NHEJ)and HR.NHEJ is the main repair pathway of DSB in eukaryotes and play roles in any cell cycle.It can complete the rapid repair of DSB by directly connecting the ends of DNA break.In the process of ligation,it is necessary to process the DNA ends,which frequently leads to the insertion or deletion of several bases(indel)at the junction,resulting in mutation of the gene.Thus,repairing of DSB by the error-prone NHEJ pathway and producing indel has become the simplest way to inactivate genes in eukaryotic cells.In addition,DSB can also be repaired by HR,which requires the introduction of a DNA repair template with homologous sequences,and homologous exchange with the targeted DNA region by recombinase to complete the repair of the DSB.In prokaryotes,due to the lack of research on NHEJ system,CRISPR-Cas editing the genome mainly through HR pathway.In this process,in addition to construct the CRISPR sites,the construction of corresponding DNA repair templates still required.The whole process is time consuming and laborious,and limits the application of CRISPR-Cas system in multiple genes,especially high-throughput genome editing.In recent years,with the development of functional genomics and DNA synthesis,high-throughput genome editing has attracted widespread attention,through genomic-scale high-throughput gene editing,fluctuating the gene expression,combined with genomics,transcriptomics,metabolomics and other omics analysis methods,can performe gene function analysis,optimization of metabolic flux,and construction of microbial cell factories more efficiently.In this thesis,we first analyzed the distribution of the typical component Ku protein in NHEJ system in prokaryotes through the protein domain family database Pfam and the protein comprehensive database InterPro.It was found that 9726 proteins have typical Prok_Ku domain,mainly distributed in Acidobacteria,Bacteroidetes,Verrucomicrobia,Proteobacteria,Actinobacteria,among them,Mycobacterium tuberculosis in actinobacteria,as a typical representative of the prokaryotic NHEJ system,has been performed many in-depth studies.We focus on a class of microorganisms that are also derived from actinomycetes and are able to tolerate very strong ionizing radiation,and analysis of the components of NHEJ system in Kineococcus radiotolerans.The ATP-dependent DNA ligase unique to the NHEJ system was found in this anti-radiation strain,indicating there is a high probability that the NHEJ repair system gives it the superior resistance to ionizing radiation.The effects of NHEJ systems in Escherichia coli DSB repair were explored by electroporation of linear plasmid DNA.All of the three different sources of NHEJ systems improved the ability of E.coli to repair DSB.Among them,we found that MTNHEJ(M.tuberculosis source)system has the most advantages in DSB repair efficiency and fidelity.The repair efficiency of intracellular DSB in E.coli was increased tenfold.We also evaluated the role of the Mt-Ku and Mt-LigD,the two components of MTNHEJ system,in the DSB repair process,and found that the lack of both will significantly reduce the repair efficiency of the DSB,and the fidelity of DSB repair will also decrease.Only in the presence of both Mt-Ku and Mt-LigD,the MTNHEJ system can fully play the role of DSB repair.We also summarized the types and characteristics of DNA mutations generated during DSB non-fidelity repair.It was found that there was a 50%probability of MTNHEJ system caused the deletion of DNA fragments of different lengths at the DSB end after ligation,which leaded to the inactivation of the gene.This established the foundation for us to develop prokaryotic gene editing tools based on the NHEJ repair system.Subsequently,we learn from the eukaryotic CRISPR-Cas9 gene editing strategy,combined the CRISPR-Cas9 with NHEJ system,developed a novel gene mutation technique that independent of DNA repair template in E.coli(CRISPR-Cas9 assisted Non-Homologous End-Joining,CA-NHEJ),which is pioneered in prokaryotic microorganism.Using the E.coli galactosidase gene as a reporter gene,we characterized and optimized the efficiency and mutation rate of this method.By knocking out recA to avoid intracellular HR and using sgRNA express CRISPR sites to avoid the appearance of repetitive sequences,the gene mutation efficiency of CA-NHEJ system was improved significantly.At the same time,gene editing with different sgRNA sites showed that CA-NHEJ can effectively introduce mutations in different genes and there is no preference for sgRNA sites.In addition,we achieved the knockout of large DNA fragments on the genome by simultaneously expressing two sgRNA sites,demonstrating the potential of this method for genome reduction.Finally,we tried to mutate genes in Pseudomonas using CA-NHEJ system,and successfully obtained the mutants that inactivated pyrF and upp genes,demonstrating the prospect of applying CA-NHEJ in various microorganisms.The CA-NHEJ system we developed is superior to traditional HR-based gene knockout technology in terms of simple operation,short experimental period,no requirement of DNA repair template and selective screening markers,and is very suitable for high-throughput gene deletion.Therefore,we improved the efficiency and mutation rate of the CA-NHEJ system,and proposed CA-NHEJ 2.0 and CA-NHEJ 3.0 gene mutation system.Among them,the CA-NHEJ 2.0 system achieved inducible and efficient genomic DNA fragment deletion through the application of tightly inducible tetracycline promoter and Cas9-Cm fusion protein expression.Combined with DNA chip synthesis,a total of 11024 sgRNA sites covering the genome of the E.coli were designed and synthesized,and genome-scale high-throughput gene deletions were performed using CA-NHEJ 2.0 system.Through screening of the mutant library by high temperature culture conditions,we not only found the key gene miaA that necessary for high temperature growth of cells,but also found that the secret phage CP4-6 in the E.coli genome is likely to be related to the high temperature environment survival of the cells.Through the optimization of promoter and replicon,CA-NHEJ 3.0 integrated all components of CA-NHEJ onto a single plasmid,and realized inducible gene deletion.By introducing a sacB-beased plasmid removal strategy,the CA-NHEJ plasmid in the completed mutant cells can be efficiently removed,and rapid genome iterative deletion is achieved.Finally,in order to overcome the interference caused by the null plasmid in the sgRNA library during the high-throughput gene deletion process of CA-NHEJ 3.0,we constructed a SOS response monitoring system by using the SOS promoter pcda to express green fluorescent protein(sfGFP).Individual level of cells DSB monitoring was achieved by flow cytometry analysis of the fluorescent intensity of different cells,and a new screening method for mutant strains was proposed,which established a foundation for the subsequent application of CA-NHEJ to microbial genome streamlining.In summary,the NHEJ-based gene deletion strategy proposed in this thesis is a powerful complement to the prokaryotic microbial gene editing tool,which effectively improves the efficiency of gene mutation in E.coli.At the same time,the high-throughput gene mutation strategy based on CA-NHEJ can rapidly analyze the correspondence between microbial genotypes and phenotypes on genome scale,and explore the potential target sites of microbial metabolic engineering,which has great significance for functional genomics research and microbial cell factory construction.Finally,CA-NHEJ can delete large DNA fragments without any DNA repair template that has a vast application prospect in the fields of genome evolution and streamlining.