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Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus

Overview of attention for article published in Microbial Cell Factories, August 2017
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Title
Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus
Published in
Microbial Cell Factories, August 2017
DOI 10.1186/s12934-017-0758-x
Pubmed ID
Authors

Ying-jie Yang, Ye Wang, Zhi-feng Li, Ya Gong, Peng Zhang, Wen-chao Hu, Duo-hong Sheng, Yue-zhong Li

Abstract

The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 50 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 50 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 7 14%
Student > Bachelor 7 14%
Researcher 6 12%
Student > Master 5 10%
Student > Doctoral Student 4 8%
Other 10 20%
Unknown 11 22%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 18 36%
Agricultural and Biological Sciences 14 28%
Immunology and Microbiology 2 4%
Chemistry 2 4%
Computer Science 1 2%
Other 2 4%
Unknown 11 22%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 06 February 2018.
All research outputs
#10,502,208
of 13,786,654 outputs
Outputs from Microbial Cell Factories
#650
of 996 outputs
Outputs of similar age
#181,571
of 268,024 outputs
Outputs of similar age from Microbial Cell Factories
#1
of 1 outputs
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