PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

Abstract

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.

Targeted Nucleotide Editing Technologies for Microbial Metabolic Engineering

“…Nevertheless, the CRISPR/Cas9 technology has been exploited for a broad range of bacteria and algae. Examples include Escherichia coli , Streptococcus pneumoniae , Bacillus subtilis , B. smithii , Streptomyces spp., Lactobacillus reuteri , L. casei , Clostridium beijerinckii , C. ljungdahlii , C. acetobutylicum , C. cellulolyticum , Pseudomonas putida , Synechococcus elongatus , Actinoplanes sp., Corynebacterium glutamicum , Staphylococcus aureus , and Myxococcus xanthus . These reports have demonstrated that the CRISPR/Cas9 system enables the easy and efficient establishment of point mutations, indels, gene replacements, large gene integrations, and large chromosomal deletions at target genomic loci.…”

Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

“…For the second fusion, kan cassette and sacB cassette from the same genomic DNA of MGL1 M were used, named as KS [14]. For the third fusion, the temples for FAR (Fatty acyl-CoA reductase) and cat-sacB are from genomic DNA of MGFARINS and MGKA, respectively, named as FS [7]. Plasmids pMD™18-T Vector, pZS13S, pEASY-Blunt were used to conserve the three fusion fragments EG, KS and FS.…”

“…The constructed pMD-KS was used for seamless deletion of poxB in E. coli strain MGL1 ( Fig. 4) according to our developed method [7]. The kan-sacB cassette was amplified with M13 primers (Table S1), and then, the products were digested with Dpn I and purified.…”

“…Then, recombinants were selected from Luria-Bertani (LB) agar plate containing kanamycin (30 mg/L). Seamless excision of the selectable marker was achieved using sucrose [7].…”

“…This technology has been improved and developed for multiple applications thereafter [4][5][6]. However, the use of traditional fusion PCR can be tedious and timeconsuming [7]. It not only calls for three PCRs to facilitate fusion of two fragments, but also requires complicated operational conditions such as adjustment of PCR conditions and cleanliness of the initial templates [8].…”