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CRISPR/Cas9-Based Counterselection Boosts Recombineering Efficiency in Pseudomonas putida.

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Summary

This study merges single-stranded DNA recombineering with CRISPR/Cas9 technology in Pseudomonas putida to efficiently generate genetic modifications. This combined approach overcomes low mutant isolation frequencies, enabling easier genome engineering.

Keywords:
CRISPR/Cas9crRNAescapersgenome engineeringspacerssDNA recombineering

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering
  • Genomics

Background:

  • Single-stranded DNA (ssDNA) recombineering facilitates genetic manipulation in Pseudomonas putida KT2440.
  • Existing ssDNA recombineering methods yield low frequencies of desired modifications, complicating the isolation of mutants lacking selectable phenotypes.
  • Efficient genetic engineering of Pseudomonas putida is crucial for its application as an environmental platform strain.

Purpose of the Study:

  • To develop a novel method for efficient isolation of genetically modified Pseudomonas putida.
  • To overcome the limitations of low-frequency mutant generation in ssDNA recombineering.
  • To enhance the genome engineering capabilities of Pseudomonas putida and potentially other Gram-negative bacteria.

Main Methods:

  • Integration of CRISPR/Cas9 technology with ssDNA recombineering in Pseudomonas putida.
  • Development of a system utilizing CRISPR/Cas9 for the efficient killing of unmodified cells, enabling non-phenotypic selection.
  • Testing the system's efficacy on various genomic modifications, including single nucleotide substitutions, gene deletions, large DNA fragment removal (flagellar cluster), and simultaneous gene deletions.

Main Results:

  • The combined ssDNA recombineering and CRISPR/Cas9 system significantly increased the frequency of desired mutants.
  • CRISPR/Cas9 targeting of non-modified wild-type genomic sequences effectively enriched for bacteria bearing intended mutations.
  • Successful introduction of diverse genetic modifications, from point mutations to large DNA fragment deletions, was demonstrated.

Conclusions:

  • The merged ssDNA recombineering and CRISPR/Cas9 system provides a powerful tool for efficient genome engineering in Pseudomonas putida.
  • This approach overcomes challenges associated with isolating mutants with non-conspicuous phenotypes.
  • The developed technology expands the genetic engineering possibilities for this environmental bacterium and may be applicable to other Gram-negative species.