Bacterial Genome Editing

The lambda Red recombineering system is a widely used tool in bacterial genome engineering. Derived from the lambda bacteriophage, this system significantly enhances homologous recombination between a foreign DNA fragment and the host genome. It consists of three primary components that work together to facilitate homologous recombination (Figure 2).

Figure 2. Key components of the lambda Red recombineering system. Exo, a 5' to 3' exonuclease, processes double-stranded DNA (dsDNA); Beta, a single-stranded DNA (ssDNA) binding protein, facilitates strand annealing; Gam, prevents host cell nucleases from degrading the linear DNA fragments introduced into the bacterial cell.

The lambda Red genes are typically expressed prior to the introduction of donor DNA. One approach is to use a plasmid encoding these components. Alternatively, bacterial strains with stable integration of the lambda Red genes are widely used for recombineering. The donor DNA, containing the desired genetic modification, is introduced into bacterial cells as either linear double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). This template often includes a selectable marker, such as an antibiotic resistance gene. The Red proteins— Exo, Beta, and Gam— facilitate homologous recombination between the donor DNA and the target sequences. Following recombination, selection methods such as drug selection followed by colony PCR are typically used to isolate cells carrying the intended modification.

CRISPR/Cas9 is a versatile genome-editing tool used across various organisms, including bacteria. In many bacterial species, such as E. coli, CRISPR-induced double-strand breaks (DSBs) are lethal due to the limited efficiency of the non-homologous end joining (NHEJ) repair pathway. This feature enables CRISPR to serve as a robust counterselection method without the need for additional selection markers.

Figure 3. Schematic representation of CRISPR editing in bacteria to create point mutations. The plasmid encoding Cas9 and the recombinases is first introduced into bacterial cells, followed by the induction of lambda Red protein expression. Subsequently, the gRNA plasmid and donor DNA are introduced into cells expressing Cas9 and the recombinases. If homologous recombination fails, the DSBs induced by Cas9 result in cell death (right). Alternatively, cells survive only if homologous recombination occurs between the donor DNA and the target genomic sequence, leading to the desired point mutations (left).

Suicide plasmids contain the homologous sequence harboring the desired modification and are designed to replicate only in a permissive host or under certain conditions. This is achieved through mechanisms such as counter-selectable markers (e.g., the sacB gene, which confers sucrose sensitivity) and conditional replication systems (e.g., temperature-sensitive origins of replication).

Figure 4. Schematic representation of gene knockin using suicide plasmids with a temperature-sensitive origin of replication. Homologous recombination integrates the gene of interest (GOI) and selection marker into the target locus. Colonies are selected with antibiotics and then cultured at the non-permissive temperature to prevent plasmid replication. A second recombination event results in either stable knockin or reversion to the original genome. Desired knock-in colonies are selected and confirmed by PCR.

Figure 5. CRISPR/Cas9 mediated E. coli genome editing. (A) Generation of a fragment deletion in the E. coli genome using the CRISPR/Cas9 system in combination with induction of the lambda Red proteins. (B) PCR of the target region had the expected amplicon size of ~3,900 bp in unedited cells (lane 1), but deletion using CRISPR-mediated knockout results in an amplicon size of approximately 1,800 bp (lane 2), exhibiting the expected ~2,100 bp deletion.