Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus
| Autor: | Wen-chao Hu, Yingjie Yang, Ye Wang, Yue-zhong Li, Duo-hong Sheng, Peng Zhang, Ya Gong, Zhi-feng Li |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Myxococcus xanthus 030106 microbiology Mutant lcsh:QR1-502 Bioengineering Computational biology Spacer sequence Applied Microbiology and Biotechnology Genome lcsh:Microbiology 03 medical and health sciences RNA Transfer Genome editing CRISPR Deletion of large genome fragments Free energy CRISPR/Cas9 Gene Sequence Deletion Genetics Base Sequence biology Cas9 Research biology.organism_classification 030104 developmental biology Genes Bacterial Epothilones Multigene Family On-target cleavage efficiency CRISPR-Cas Systems GC-content Plasmids RNA Guide Kinetoplastida Biosynthetic gene clusters for secondary metabolites Biotechnology |
| Zdroj: | Microbial Cell Factories, Vol 16, Iss 1, Pp 1-15 (2017) Microbial Cell Factories |
| ISSN: | 1475-2859 |
| DOI: | 10.1186/s12934-017-0758-x |
| Popis: | Background 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0758-x) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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