PCRISPomyces-2

Use

This plasmid can be used for precise CRISPR/Cas9-mediated genome engineering of Streptomyces strains. To our knowledge it has been successfully used in S. albidoflavus, S. coelicolor, S. formicae, S. lividans and S. venezuelae. Mutations have ranged from 1-100kbp deletions, precise codon changes to alter amino acids and insertions to add Flag-tags to proteins encoded at their native loci.

The pCRISPomyces2 paper

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