Oligonucleotide Recombination Engineering And CRISPR-Cas9 Are Combined For Use In E. Coli Genome Editing

Recombineering(recombinant-mediated genetic engineering)is usually refers to the use of ? phage-derived recombinases to catalyze homologous recombination between DNAs.Therefore,it is also referred to as ? Red recombination engineering.Because of its simple operation,rapidity and high efficiency,recombineering has become a commonly used genetic engineering method for DNA cloning and DNA modification.Single-stranded oligonucleotide-mediated recombineering has the advantage of achieving simultaneous editing of multiple sites in the bacterial genome,rendering it a highly efficient tool in gene editing which is much better than traditional recombination engineering techniques.CRISPR/Cas system,in which the CRISPR is abbreviation of cluster of regularly spaced short palindromic repeats and Cas is CRISPR-associated protein,is rapidly becoming an essential gene editing strategy ranging from bacteria to human cell lines.CRISPR/Cas system is characterized by directed repeats of nearly the same sequence interspersed by spacers which are of different sequences.The spacers are found to share the same sequence of many phage DNAs or mobile DNA elements that can invade bacteria.About 50%of bacteria and 80%of archaea use CRISPR-Cas system as the immune system to protect against viruses and other invading nucleic acids.The Type ?Cas9 protein from Steotococcus pyogenes SF370 is most thoroughly studied and most widely used in the CRISPR/Cas gene editing system.Because the system is able to cleave DNA,and the specificity of its cleavage sequence can be programmed by the sequence of crRNA,CRISPR/Cas can bused for the gene editing of any sequence.This thesis focuses on the combination of oligonucleotid-mediated recombineering and CRISPR-Cas9 system to the gene editing of Escherichia coli.Oligonucleotid-mediated recombineering and CRISPR-Cas9 system was firstly used in E.coli gene editing in 2003.The E.coli primase DnaG is involved in DNA replication and its Q576A and K580A point mutants were reported to increase the DNA recombination efficiency;however,neither Q576A K580A double mutants were obtained nor direct recombineering efficiency comparison was reported.We applied the Oligonucleotide engineering and CRISPR-Cas9 fro DnaG mutation.Firstly,we constructed serial cas9 expression vectors and sgRNA expression vectors including high copy number pMB1 replicon-based cas9 expression plasmid,low-copy number p15A replicon-based cas9 expression plasmid,high copy number pUC replicon-based sgRNA expression plasmid and low-copy pBBR1 replicon-based sgRNA expression plasmid.After performing transformation efficiency comparison,the system consisting of p15A replicon-based cas9 expression plasmid and pUC replicon-based sgRNA expression plasmid was chosen for further study.The pUC replicon-based sgRNA expression plasmid was designed to contain I-Sce1 homing endonuclease and its cleavage sites,making it a selef-cleavage vector.Co-transformation of mutagenic oligonucletide and gRNA expression plasmid into recombineering proficient and CRISPR/Cas9 proficient E.coli cells and the resulting transformants were genotyped via PCR and DNA sequencing.Q576A and K580A single mutants were successfully obtained,which named E.coli LS3513 and E.coli LS3516.These single mutations efficiencies respectively reached to 40%and 100%.The elimination efficiencies of sgRNA expression plasmids in E.coli LS3513 and E.coli LS3516 were respectively 100%and 44%;however no Q5 76A K5 80A double mutant was obtained though various attempts.Instead a structurally similar hydrophobic amino acid valine(Val),which named E.coli LS3519V.The mutation efficiency reached 5%and the elimination efficiency of sgRNA expression plasmid is 70%;besides threonine(T)was mostly frequently obtained,which named E.coli LS3519T.The mutation efficiency reaches 73%and the elimination efficiency of the sgRNA expression plasmid is 46%.The second part of the study is the engineering of enzymes involved in N-acetyl-D-neuraminic acid(Neu5Ac)biosynthesis.Found at the ends of biological macromolecules can participate in many physiological reactions in the organism,Neu5Ac is the mostly-valued intermediates used in anti-fu drug zanamivir.ppsA and csrB are the two genes that are involved in biosynthesis of phosphoenolpyruvate that are precursors of Neu5Ac.By coupling oligonucleotide recombineeing and CRISPR-Cas9,T7 promoter was engineered to replace the promoter of ppsA and csrB genes.We obtained a strain that a single insertion of the ppsA gene region into the T7 promoter,which named E.coli LS3561.The mutation efficiency was 5%.A strain named E.coli LS3571 whose csrB gene region was inserted into the T7 promoter with a mutation efficiency of 100%.A strain named E.coli LS3581 whose ppsA and csrB gene regions are both inserted the T7 promoter with the mutation efficiency of 10%.The elimination efficiencies of the sgRNA expression plasmid in E.coli LS3561,E.coli LS3571,and E.coli LS3581 respectively up to 100%,2%and 84%.In addition,we constructed the self-clearing vector pLS3550 with p15A plasmid backbone and applied it to the elimination of cas9 expression plasmid pLS3535.The elimination of cas9 expression plasmid was successfully eliminated and the efficiency reached 21%,24%and 41%,respectively.p15A replicon-based self-cleavage vector pLS3550 was constructed and used to successfully eliminate the p15A replicon-based cas9-expression vector.The elimination of cas9 expression plasmid elimination was successful,with efficiencies of 21%,24%and 41%,respectively.The engineered strains have the potential for Neu5 Ac enzymatic catalysis.