CRISPR-Assisted Multiplex Base Editing System in Pseudomonas putida KT2440

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Pseudomonas putida KT2440 is a key environmental bacterium with applications in synthetic biology, metabolic engineering, and biodegradation.
• Existing genome editing methods for P. putida are often laborious and inefficient, relying on heterologous repair proteins and donor DNA templates.

Purpose of the Study:

• To develop an efficient and convenient base editing system for Pseudomonas species.
• To enable gene inactivation and multiplex editing without requiring DNA strand breaks or donor DNA templates.

Main Methods:

• Established a cytosine base editing system using cytidine deaminase (APOBEC1), enhanced specificity Cas9 nickase (eSpCas9ppD10A), and uracil DNA glycosylase inhibitor (UGI).
• Engineered an eSpCas9ppD10A-NG variant to expand editable sites and modified the APOBEC1 domain to narrow the editable window.
• Applied the system for gene inactivation and multiplex base editing in various Pseudomonas species.

Main Results:

• The base editor efficiently converts C-G to T-A without DNA breaks or donor templates.
• Achieved gene inactivation efficiencies of 25-100% in multiple Pseudomonas species.
• Demonstrated multiplex base editing in double (90-100% efficiency) and triple loci (25-35% efficiency).

Conclusions:

• The developed base editing system is fast, convenient, and universal for Pseudomonas species.
• This system significantly accelerates research in synthetic biology, metabolic engineering, and biodegradation using P. putida and other Pseudomonas species.