A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy

Wang, Jichao; Sui, Xinyue; Ding, Yamei; Fu, Yingxin; Feng, Xinjun; Liu, Min; Zhang, Youming; Xian, Ming; Zhao, Guang

doi:10.1093/nar/gkaa1141

Cited by 34 publications

(17 citation statements)

References 49 publications

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“…This entire process was completed in 8 days. In comparison, using the mature CRISPR-Cas9 system to edit 14 sites, according to the latest reported iterative genome-editing method 15 to edit one site every round, requires 28 days in total, which is 3.5 times of that taken by MUCICAT. Futhermore, the workload was greatly reduced using MUCICAT.…”

Section: Discussionmentioning

confidence: 99%

“…14 In Escherichia coli, at least 2 days were needed for one copy of chromosomal integration. 15 Alternatively, the bacteriophage Mu-driven transposition system, in principle, allows for the integration/amplification of target genes in the chromosomes, but the integration sites are random. 16 Recently discovered transposon-encoded CRISPR-Cas systems, including I-F3 and V-K subtypes, [17][18][19] enable genomic site-specific DNA integration without introducing DSB, which means they are independent of HR.…”

Section: Introductionmentioning

confidence: 99%

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Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPRassociated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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“…We first constructed the genome integration strain GF-5 (Additional file 1 : Table S2), which is a counterpart of GF-1. In brief, taking advantage of a fast and robust iterative genome-editing CRISPR/Cas9 system based on the Rock-Paper-Scissors strategy [ 35 ], the gene encoding RrUGT3 was successfully integrated into the genome of E. coli BL21(DE3) at the ushA gene locus (in-frame deletion), knockout of which was reported to increase the supply of UDPG [ 15 ]. Upon feeding of 1 , GF-5 exhibited a similar activity (yield 99.8%) as the corresponding plasmid-based strain GF-1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The CRISPR/Cas9 technology was adopted for E. coli genome editing, which was carried out based on the reports of Li et al [ 68 ] and Wang et al [ 35 ] with some modifications. The ushA gene was chosen as a locus for heterogeneous gene integration.…”

Section: Methodsmentioning

confidence: 99%

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity

et al. 2022

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Background Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides. Results In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides. Conclusions Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

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“…Iterative genome editing (IGE) requires to efficiently cure the genome editing plasmid, and targeting replicons or resistance genes of the plasmid using a gRNA has been employed for this purpose [14,[16][17][18][19]. In this study, we constructed an all-in-one plasmid that utilizes CRISPR-Cas9 system for IGE in B. subtilis.…”

Section: Design and Construction Of An All-in-one Plasmid Crispr-cas9...mentioning

confidence: 99%

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

et al. 2022

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Background Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. Results To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. Conclusions We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

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A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy

Cited by 34 publications

References 49 publications

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

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