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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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Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria.
Chenhao Li1,2, Longfei Wang1, Leland J Cseke1
1Division of Plant Science and Technology, Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.
Communications Biology
|January 16, 2023
Summary
New CRISPR base editors efficiently modify genes in diverse plant pathogens like Xanthomonas and Pseudomonas. This tool enhances genetic analysis of bacterial diseases, with a modified version reducing unintended mutations for improved accuracy.
Area of Science:
- Molecular Biology
- Plant Pathology
- Microbial Genetics
Background:
- Phytopathogenic bacteria significantly impact crop yields, necessitating advanced genetic tools for understanding disease mechanisms.
- CRISPR genome editing offers powerful applications in prokaryotes, but host-specific adaptations are often required.
- Existing CRISPR systems face limitations in broad applicability across diverse bacterial species.
Purpose of the Study:
- To develop novel CRISPR-based base editors (cytosine base editors, CBEs) for broad-spectrum genome editing in phytopathogenic bacteria.
- To evaluate the efficiency and host range of CBEs utilizing dCas9 and nCas9 nucleases fused with cytosine deaminase.
- To assess the potential for multiplexed gene editing and minimize off-target mutations in bacterial pathogens.
Main Methods:
- Engineered CRISPR-dCas9 and nCas9 deaminase fusion proteins (CBEs) with cytosine deaminase (CDA1).
- Utilized different promoters, including the RecA promoter (RecAp), for controlling CBE expression.
- Integrated CBE constructs into broad-host-range plasmid pHM1 for delivery into various bacterial strains.
- Performed multiplexed guide RNA strategies for simultaneous editing of multiple genes.
- Conducted whole-genome sequencing to identify on-target and off-target mutations.
Main Results:
- The RecA promoter-driven CBE (CBERecAp) achieved nearly 100% target modification efficiency.
- CBERecAp demonstrated efficacy in editing Xanthomonas, Pseudomonas, Erwinia, and Agrobacterium strains.
- CRISPR-based nCas9 editors expanded the editing window and increased modification rates in Pseudomonas.
- Multiplexing enabled the simultaneous editing of up to four target genes.
- A modified CBE (CBERecAp-A) significantly reduced off-target mutations compared to the initial CBE.
Conclusions:
- Developed CRISPR-dCas9 and nCas9 cytosine base editors are effective tools for broad-spectrum genome editing in phytopathogenic bacteria.
- The RecA promoter enhances editing efficiency, and the pHM1 plasmid facilitates delivery across diverse bacterial genera.
- Multiplexing capabilities allow for efficient modification of multiple genes, accelerating genetic studies.
- The modified CBERecAp-A offers an improved, lower-off-target editing system for phytopathogenic bacteria research.
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