Evidence map›Paper›PMID 35643496›Full record

ArticleMicrobial cell factories2022

Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02.

Qinglong Xin, Yudan Chen, Qianlin Chen, Bin Wang, Li Pan

Open access · goldAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Microbial cell factories, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.

0numbers the graph read from it
0cells of the map it votes in
8citing papers in PubMed
1.5field-weighted citation impact, top 18% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

8 citing papers in PubMed, 22 citations in OpenAlex.

  1. Article
  2. Review
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  7. Microorganisms · 2023
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4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

5 authors at 1 institution in 1 country.

Qinglong XinSchool of Biology and Biological Engineering, Guangzhou Higher Education Mega Centre, South China University of Technology, Panyu District, Guangzhou, 510006, Guangdong, People's Republic of China.
Yudan ChenSchool of Biology and Biological Engineering, Guangzhou Higher Education Mega Centre, South China University of Technology, Panyu District, Guangzhou, 510006, Guangdong, People's Republic of China.
Qianlin ChenSchool of Biology and Biological Engineering, Guangzhou Higher Education Mega Centre, South China University of Technology, Panyu District, Guangzhou, 510006, Guangdong, People's Republic of China.
Bin WangSchool of Biology and Biological Engineering, Guangzhou Higher Education Mega Centre, South China University of Technology, Panyu District, Guangzhou, 510006, Guangdong, People's Republic of China. btbinwang@scut.edu.cn.
Li PanSchool of Biology and Biological Engineering, Guangzhou Higher Education Mega Centre, South China University of Technology, Panyu District, Guangzhou, 510006, Guangdong, People's Republic of China. btlipan@scut.edu.cn.
South China University of Technology · CN

Funding

Core Technology Project of Foshan City 1920001000824National Key Research and Development Program of China 2021YFC2100200Research and Development Plan in Key Areas of Guangdong Province 2018B020205002Research and Development Plan in Key Areas of Guangdong Province 2020B020226007
6 · The paper itself

Abstract

backgroundBacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus. This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02.

resultsIn this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes (aprE, nprE, wprA, and bamHIR) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain.

conclusionsThe CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.

Indexed as

Bacillus amyloliquefaciensCRISPR-Cas Systemsalpha-AmylasesDNA Restriction-Modification EnzymesGene Editingalpha-AmylasesDNA Restriction-Modification EnzymesB. amyloliquefaciensBase editingCRISPR/Cas9n-AIDα-amylase

Identifiers

PMID35643496
PMCPMC9148480
OpenAlexW4281648324

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LicenceCC BY
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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.