Escherichia coli is an important model organism. Puinte Bio has mastered mature E. coli gene editing technologies and can provide clients with comprehensive E. coli gene editing services, including gene knockout, gene knock-in, and site-directed mutagenesis. The core technologies include λ-Red homologous recombination gene editing, CRISPR gene editing, and scarless gene editing that combines the advantages of both methods.

Red homologous recombination E. coli gene editing services:

The Red homologous recombination system is a recombination system derived from the lambda phage. This system expresses the three proteins Gam, Exo, and Beta on the pKD46 vector, allowing foreign DNA to be homologously recombined into the E. coli genome. The system is mature and efficient, enabling gene knock-in and knock-out. Our company has incorporated the Cre-loxP system into the Red recombination system to remove the inserted selectable marker gene, essentially achieving scarless editing.

CRISPR Cas9 E. coli Gene Editing Services:

CRISPR Cas9 precisely cuts genomic DNA, which is then repaired by the Red recombinase. E. coli that fail to successfully repair die due to genomic DNA breakage, while individuals that are successfully repaired—that is, successfully edited—survive. The CRISPR Cas9 system can achieve truly scarless gene editing. Compared to the Red homologous recombination system, the CRISPR Cas9 system has a significant advantage in precise site-specific mutagenesis due to its unique mechanism for screening positive clones without tags.

iStop E. coli Gene Editing Services:

iStop technology is an application of CRISPR CBE technology. This technology does not require double-strand cutting of DNA and does not cause cell death. It guides enzymes to specific locations in the E. coli genome through CRISPR sgRNA, causing C>T mutations at the target site. This converts several codons such as CAA, CGA, and CAG into stop codons, leading to premature termination of gene translation. The principle of gene knockout with this technology is that incomplete gene products result in loss of gene function. Its advantages lie in making only minimal changes to the genome, causing very little disturbance to the organism. Moreover, it does not require a recombination template, has a short construction cycle, and is cost-effective.

Service Advantages

▶Extensive Experience: With rich experience in E. coli gene editing, our experienced team can help you quickly construct E. coli mutants;

▶Three Experimental Options: Choose any of the three experimental schemes;

▶One-Stop Solution: We provide comprehensive upstream and downstream services, including E. coli gene knockout and complementation validation experiments;

▶Quality Assurance: All gene editing sites are sequenced and verified to ensure product quality.

Service Process

Services	Red homologous recombination	CRISPR Cas9 gene editing	iStop gene editing
Gene editing efficiency	High	Relatively high, with certain requirements for targets and genes	High
Whether a recombinant template is needed	Need	Need	Not needed
Cost	Higher	Higher	Lower
Edit without leaving a trace	No	Yes	Yes
Scope of application	Gene knockout and knock-in	Knockout, site-directed mutation	Knockout

Article Case

Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria

academic journal：International Journal of Biological Macromolecules

SSB/CRISPR-Cas9 non-homologous and homologous recombination gene editing of the upp gene in E. coli MG1655.

(A) Results of plating different transformation systems. (B) PCR verification of non-homologous recombination gene editing transformants. WT, E. coli MG1655; 1–15, randomly selected transformants; uppΔ(43-267), upp gene with a deletion of 43 to 267 base pairs. (C) PCR verification of homologous recombination gene knockout transformants using ssODN donor. Δupp, knockout of the upp gene. (D) PCR verification of homologous recombination gene knockout transformants using tetRdsODN donor. Δupp::tetR, upp gene replaced by tetR. (E) Editing efficiencies of three transformation systems for the E. coli upp gene. Data are presented as mean ±standard deviation from nine independent experiments. Based on one-way ANOVA, **p < 0.01, ****p < 0.0001.

Applicable Fields

▶Basic Research and Functional Genomics: Study the function of individual genes, construct whole-genome sgRNA libraries, and systematically investigate the relationships between genes and phenotypes; construct nutrient-deficient strains (by knocking out essential genes) for coupled screening.

▶Industrial Biomanufacturing and Metabolic Engineering: Knock out competitive or by-product pathways to optimize metabolic flux; knock out non-essential structural genes to save cellular resources; construct nutrient-deficient coupled systems to accelerate strain evolution.

▶Biomedicine and Therapeutic Applications: Engineer probiotics (such as EcN) as platforms for therapeutic protein expression; construct attenuated vaccine strains; develop "live biopharmaceuticals," such as knocking out genes and inserting the `ttr` cluster to enhance colonization.

▶Strains and Model Construction: Build gene engineering systems without antibiotic resistance markers; prepare specific strains for feeding model organisms (such as nematodes).

Gene editing by ssb_crispr-cas9 ribonucleoprotein in bacteria.pdf