LEUpOUT and HIS/FLP systems for marker/CRISPR recycling.

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LEUpOUT and HIS/FLP systems for marker/CRISPR recycling. LEUpOUT and HIS/FLP systems for marker/CRISPR recycling. (A) The LEUpOUT-based CRISPR systems are integrated within the LEU2 ORF and disrupt LEU2 expression. The resulting direct repeats (indicated by the EU portions of the disrupted LEU2 ORF) enable spontaneous excision of the CRISPR components via homologous recombination (LEUpOUT). LEU+/NAT− transformants can be selected on medium lacking leucine. US_LEU2 and DS_LEU2 represent genomic sequences 5′ and 3′ to the LEU2 ORF, respectively. (B) The HIS/FLP system is integrated at the HIS1 locus, replacing one allele of the HIS1 ORF. CRISPR/NAT excision is mediated by the maltose-inducible FLP recombinase, leaving a single FLP recombinase target (FRT) site in place of the HIS1 ORF. US_HIS1 and DS_HIS1 represent genomic sequences 5′ and 3′ to the HIS1 ORF, respectively. Namkha Nguyen et al. mSphere 2017; doi:10.1128/mSphereDirect.00149-17