Development Of New Techniques For Genome Editing,Directed Evolution And Protein-DNA Interaction Determination

Genomic DNA is the biggest and most important molecule for all of livings on earth.It carries the genetic information and directs the biosynthesis of RNA and proteins,which regulate almost all life processes and perform a diverse range of functions for the cell.Genome-related researches,such as genome sequencing,targeted genome editing,targeted locus mutagenesis,the mapping of global regulatory networks,as well as the de novo design and synthesis of genome,have increased our understanding of genomes,and empowered us the ability to optimize functions or even create new functions of life.On the other hand,the complexity of life on this planet is by no means designed and engineered by some rational forces,but by a simple and elegant power:evolution.The continuous and incremental introduction of mutations or variations in the genetic code allows new functions to develop from old ones or entirely parallel ones to appeal.Directed Evolution,the technology of learning and harnessing this natural process,can also endow us the aptitude to understand and modify life.Combining the background with the needs of our laboratory,this thesis presents three topics related to targeted locus mutagenesis,mapping of genomic regulatory networks and genome manipulating,which are:1)random mutation of specific regions of the genome using chimera of cytosine deaminase AID protein and T7 RNA polymerase;2)identifying genome-wide targets of proteins(here transcription factors,TFs)in vivo by fusing them to AID,expressing the obtained fusion proteins and high-throughput sequencing;3)developing a plasmid system that can be used for gene inactivation and fusion,gene elements replacement,in-situ transcription type reporter construction and large fragments deletion in genome,based on singlecrossover alleles exchange.Subject 1:FAST-TM(Fusing AID to Specific T7 RNA polymerase for Targeted locus Mutagenesis).An important part of Directed Evolution is to create mutation libraries of target genes.AID can convert a C:G base pair to a T:A base pair in genome,T7 RNA polymerase can initiate transcription from T7 promoter in a highly-specific way,fusing AID to T7 RNA polymerase can combine the characteristics of the both.As transcription starts from the T7 promoter,the chimera repeatedly ’sliding’ on the downstream gene region,AID can leave‘mutation marks’ at the same time.As a proofof-concept,we inserted a sequence of fluorescent protein gene with T7 promoter(PT7mScarlet)into Pseudomonas aeruginosa genome and induced expression of the fusion protein.Just as expected,fluorescence and mutations on the sequence were both observed.Then we investigated the specificity of the method by using kanamycin resistance recovery assay,the results showed that only under T7 promoter the kanS gene with inactivating point mutation was repaired to restore the resistance.High-throughput sequencing of PT7-mscarlet sequence showed that the fusion protein group could detect much higher mutation frequency across the entire region compared to the control group expressing AID protein alone.Finally,we evolved wild-type PAO1 with resistance to aminoglycoside antibiotics within 24 hours by the called FAST-TM method.Subject 2:MutID(Mutation-based protein-DNA Interaction Detection).A large variety of proteins,especially TFs,regulate gene expression by binding to specific parts of the genome.It is important to map their binding locations across the whole genome.Commonly used methods for detecting interactions between proteins and genomic DNA are based on chromatin immunoprecipitation(e.g.ChIP-seq,ChIP-chip),which requires complex operations and highly dependents on specific antibodies.Here we describe MutID,a novel technique to map genome-wide transcription factor binding sites(TFBSs)that couples a TF or a protein of interest to activation-induced cytidine deaminase and expresses the chimeric protein in vivo.TFBSs are labeled with DNA editing events and identified by whole genome sequencing.Using MutID,we have identified the genome-wide targets of LasR in Pseudomonas aeruginosa and found six promoters regulated by LasR that had never been reported before.We also studied the relationship between mutations and transcription on the promoter,and discuss the advantages and disadvantages of the MutID approach as well as the direction for improving it.Subject 3:Construction and applications of a gene manipulating tool-the pln2 plasmid system.The original aim of this project was to develop a simple and quick method for Pseudomonas aeruginosa gene knockout.To achieve this goal,we designed and built a light non-replicable plasmid pln2 for efficient integration into the Pseudomonas aeruginosa genome.When a gene needs to be inactivated,cloning of an internal fragment of the target gene(non-frameshift)into the pln2 plasmid and once single-crossover alleles exchange between genome and the obtained plasmid happened,the endogenous gene is segmented by the plasmid backbone and thus inactivated.Based on pln2,we have developed a series of plasmids with different uses for different genomic operating,including:fusion of fluorescent protein gene,replacement of gene elements(e.g.promoters,rbs),construction of in-situ transcriptional fluorescent reporters,large fragments knockout.We emphasize the work of large fragments knockout here,which involves inserting two FRT sites on both sides of the target fragment by pln2 system and inducing the expression of flipping enzyme FLP to delete the sequence between the FRT sites.We have used this method to knock out the type III secretory system gene cluster(25 kbp),the prophage DNA gene cluster(22 kbp),and further knock out the PA1184-PA1373 and PA2264-PA2452 segments,which are about 470 kbp in length.In total,8.3%of Pseudomonas aeruginosa genome sequences were deleted.