Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas.

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• CRISPR-Cas systems are powerful for bacterial genome editing but often require host factors or exogenous recombinases.
• Existing methods face limitations in efficiency and applicability across different bacterial species, especially those with less efficient homologous recombination.

Purpose of the Study:

• To develop a simple, widely applicable genome engineering tool for bacteria that mitigates constraints of existing CRISPR-Cas systems.
• To enable efficient gene editing in bacteria, including those with poor homologous recombination systems, without relying on exogenous recombinases.

Main Methods:

• Engineered a Self-splicing Intron-Based Riboswitch-Cas (SIBR-Cas) system using a mutant T4 td intron library for inducible control.
• SIBR-Cas provides delayed CRISPR-Cas counter-selection to allow time for homologous recombination-mediated editing.
• Applied SIBR-Cas to knock out genes in wild-type Escherichia coli, Pseudomonas putida, and Flavobacterium species.

Main Results:

• Successfully achieved gene knockouts in three distinct bacterial species (E. coli, P. putida, F. IR1) using SIBR-Cas.
• Demonstrated efficient genome editing without the need for exogenous recombinases, even in bacteria with poor native homologous recombination.
• SIBR-Cas offers tight and inducible control over CRISPR-Cas counter-selection, enhancing editing efficiency.

Conclusions:

• SIBR-Cas is a simple, tightly regulated, and broadly applicable genome engineering tool for diverse bacterial species, particularly non-model organisms.
• The SIBR system shows potential for broader applications in bacterial gene expression and regulation control.
• This approach overcomes key limitations of current bacterial genome editing technologies.