pCRISPomyces-2

Use

This plasmid can be used for precise CRISPR/Cas9-mediated genome engineering of Streptomyces strains¹. It is possible to introduce mutations that range from 1-100kbp deletions, precise single base codon changes to alter amino acids and insertions to add Flag-tags to proteins encoded at their native loci.

This plasmid has been successfully used for genome editing in the following organisms

• Actinoplanes sp. SE50/110²
• Streptomyces albidoflavus³
• Streptomyces coelicolor (unpulished mutagenesis performed within the group of Prof Matt Hutchings)
• Streptomyces formicae⁴
• Streptomyces lividans¹
• Streptomyces rimosus⁵
• Streptomyces roseosporus⁶
• Streptomyces showdoensis⁷
• Streptomyces venezuelae⁶
• Streptomyces viridochromogenes¹

See here for the original pCRISPomyces-2 paper from Cobb et. al¹.

Click here for the CRISPR protocol used by Matt Hutchings lab (courtesy of Rebecca Devine) - or download the PDF.

Features

• Apramycin resistance cassette
• Origin of conjugative transfer (oriT)
• Origin of replication for Escherichia coli (pBR322oriF)
• Codon optomised cas9 gene under the constitutive rpSL promoter
• LacZ cassette for golden gate cloning of guide RNA (gRNA)
• Constitutive gapdh promoter for gRNA expression
• pSG5 temparature sensitive Streptomyces origin of replication. Without selection and above 37°C the plasmid becomes unstable. This property is used to cure the plasmid from Streptomyces after successful mutagenesis.
• Xba1 site for gibson assembly of repair templates

History

The plasmid was made by Huimin Zhao's group and is available free from AddGene under MTA: click here

Map

Sequence links

AddGene under MTA: click here

References

1. Cobb R.E., Wang, Y., Zhao, H. (2014). High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synthetic Biology, 4(6), pp. 723-728. DOI: 10.1021/sb500351f
2. Wolf, T., Gren, T., Thieme, E., Wibberg, D., Zemke, T., Pühler, A., Kalinowski, J. (2016). Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System. Journal of Biotechnology, 231, pp. 122-128. DOI: 10.1016/j.jbiotec.2016.05.039.
3. McLean, T.C., Hoskisson, P.A., Seipke, R.F. (2016). Coordinate Regulation of Antimycin and Candicidin Biosynthesis. mSphere, 1(6), pp. e00305-e00316. DOI: 10.1128/mSphere.00305-16.
4. Qin, Z., Munnoch, J.T., Devine R., Holmes, N.A., Seipke, R.F., Wilkinson, K.A., Wilkinson., Hutchings, M.H. (2017). Formicamycins, antibacterial polyketides produced by Streptomyces formicae isolated from African Tetraponera plant-ants. Chemical Science, 8, pp. 3218-3227. DOI: 10.1039/c6sc04265aT
5. Jia, H., Zhang, L., Wang, T., Han, J., Tang, H., Zhang, L. (2017). Development of a CRISPR/Cas9-mediated gene-editing tool in Streptomyces rimosus. Microbiology, 163(8), pp. 1148-1155. DOI: 10.1099/mic.0.000501.
6. Zhang, M.M., Wong, F.T., Wang, Y., Luo, S., Lim, Y.H., Heng, H., Yeo, W.L., Cobb, R.E., Enghiad, B., Ang, E.L., Zhao, H. (2017). Crispr–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nature Chemical Biology, 13, pp. 607-609. DOI: 10.1038/nchembio.2341
7. Palmu, K., Rosenqvist, P., Thapa, K., Illina, Y., Siitonen, V., Baral, B., Mäkinen, J., Belogurov., Virta, P., Niemi, J., Metsä-Ketelä, M. (2017). Discovery of the Showdomycin Gene Cluster from Streptomyces showdoensis ATCC 15227 Yields Insight into the Biosynthetic Logic of C-Nucleoside Antibiotics. ACS Chemical Biology, 12(6), pp. 1472-1477. DOI: 10.1021/acschembio.7b00078.