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

• Streptomyces albidoflavus²
• Streptomyces coelicolor (unpulished mutagenesis performed within the group of Prof Matt Hutchings)
• Streptomyces formicae³
• Streptomyces lividans¹
• Streptomyces venezuelae (unpulished mutagenesis performed within the group of Prof Matt Hutchings)

See here for the original pCRISPomyces2 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

• Ampicillin 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. 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.
3. 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/c6sc04265a