Oligonucleotide Recombination Engineering And CRISPR-Cas9 Mediated Genome Editing Of Pseudomonas Putida KT2440

Receombinerring??-Red/ET technology?is a DNA cloning and modification biotechinology via phage-origin recombinase-catalyzed hmologos recombination.Receombinerring is easy to manipulate,no cloning step is involved,and no restrictions on either the restriction enzyme recongnition sites or the size of target DNA.ssDNA recombineering,which can be applied for multiplexed genome editing,is a more powerful recombineering technology.CRISPR-Cas system?Cluster of Regularly Spaced Short Palindromic Repeats and Cas protein?is an evolutionized bacteria and archaea adaptive immunity system against incoming DNAs such as phage infection,plasmid conjugation and transformation.Currently,type II CRISPR-Cas system is the most studied and most widely used genome editing system.The high efficiency and high programmability of CRISPR-Cas system make it a estremly useful genome editing tool.Pseudomonas putida KT2440 is a typical Gram-negative strain and is reapidly becoming the heterologous expression host of value-added compounds.P.putida KT2440 is also able to degrade environmental toxic compounds.These chracteritics endow P.putida KT2440 the great potential for both basic research and prarical applications.In this study,we explored P.putida KT2440 genome editing technology via the combination of ssDNA recombineering and CRISPR-Cas9.Firstly,based on ssDNA recombinering-mediated recombination efficiency comparison of the kan fragment repaired to kanamycin resistance gene kan,the best recombinase gene red? was selected.Then pLS3819 cloned with inducible expression of red? and inducible expression of cas9 was constructed.Several sgRNA promoters were tested on the ssDNA recombineering and CRISPR-Cas9-mediated mkan repair efficiency,and Pm promoter was found to be optimized for sgRNA expression.Then P.putida KT2440 endogenous genes were edited.The point mutations of pheS and upp genes were achieved with editing efficiency of 75% and 100% respectively.The upp,PP₀589,PP₃548 and PP₂064–PP₂069?621 bp,1164 bp,1518 bp and 9293 bp,respectively?gene?s?were also sucessfully deleted with the deletion efficiency of 100%,85%,70% and 3%.A trend that the large the target gene size,the less the gene deletion efficiency.However,it is still relatively easy to obtain a mutant strain..The herein established genome editing technique is convenient and shows relatively high efficiency.Lastly,a series of reported gene lacZ harboring vectors and plasmids were in an effort to estabish a P.putida KT2440 CRISPRi?CRISPR interference?system.It tured out,however,the enzymatic activity of the integrated LacZ is too low to be suitable for CRISPRi assay.Further modifications are required to investigate the P.putida KT2440 CRISPRi system.