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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
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Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing
Daniel C Volke1, Román A Martino2,3, Ekaterina Kozaeva1
1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
Nature Communications
|May 31, 2022
Summary
This study introduces a new CRISPR base editing toolset for efficient genome engineering in Gram-negative bacteria. The technology enables precise single-nucleotide changes and multiplex editing, overcoming previous limitations.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- CRISPR/Cas technologies are powerful genome engineering tools.
- Their application in non-traditional bacteria is limited by host factors and exogenous recombinases, impacting efficiency and throughput.
Purpose of the Study:
- To develop a versatile genome engineering toolset for Gram-negative bacteria.
- To overcome limitations of existing technologies for broader applicability.
Main Methods:
- Tailored a CRISPR base editor for single-nucleotide resolution (C·G → T·A) with >90% efficiency.
- Incorporated Cas6-mediated guide RNA processing for streamlined multiplex base editing (>85% efficiency).
Main Results:
- Demonstrated successful application in Pseudomonas putida, a soil bacterium.
- Engineered complex phenotypes, including single-step aromatic compound production and multi-step redox metabolism deconstruction.
Conclusions:
- The developed toolset significantly enhances genome engineering capabilities in Gram-negative bacteria.
- This approach overcomes previous constraints, enabling previously inaccessible engineering programs.
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