Genotype analysis of transient CRISPR system transformants.

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Genotype analysis of transient CRISPR system transformants. Genotype analysis of transient CRISPR system transformants. (A) PCR detection strategy for ADE2 (left) and ade2Δ::NAT (right) alleles. The 5′-end-flanking primer ADE2-fwd was used with ADE2 internal primer ADE2-rev to detect the ADE2 allele and with NAT internal primer NAT-rev to detect the ade2Δ::NAT allele. (B) Allelic content of transformants. Genomic DNA was used for PCR genotyping from 12 white transformants, 20 red transformants, the parental strain (lane P), CaCAS9-and-sgRNA plasmid pV1093 (lane C), or a no-template control (lane N). Transformants with the ADE2.1 or ADE2.2 sgRNA gene were obtained, as indicated across the top. Genotyping reactions were for ADE2, ade2Δ::NAT, the sgRNA cassette, or an internal segment of CaCAS9, as indicated to the left. (C) PCR detection strategy for FRP1 (left) and frp1Δ::ARG4 (right) alleles. The 5′-end-flanking primer FRP1-fwd was used with FRP1 internal primer FRP1-rev to detect the FRP1 allele and with CdARG4 internal primer ARG4-rev to detect the frp1Δ::CdARG4 allele. (D) Allelic contents of transformants. Genomic DNA from 8 transformants, as well as wild-type strain SC5314 (lane W), was used for PCR genotyping. Genotyping reactions were for FRP1 or frp1Δ::CdARG4, as indicated to the left of each gel image. Kyunghun Min et al. mSphere 2016; doi:10.1128/mSphere.00130-16