Strategy for marker recycling through CRISPR-Cas9-induced marker excision. Strategy for marker recycling through CRISPR-Cas9-induced marker excision. Consider.

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Strategy for marker recycling through CRISPR-Cas9-induced marker excision. Strategy for marker recycling through CRISPR-Cas9-induced marker excision. Consider a situation in which an investigator seeks to make a C. albicans strain with homozygous deletion mutations in three genes—YFG1, YFG2, and YFG3—using only two selection markers, M1 and M2. The marker cassettes, modified to include flanking direct repeats, are designated rM1r and rM2r. The three YFG genes are shown on separate blue, red, and violet chromosomes. The construction is carried out with only three transformations. For transformation 1, first a homozygous yfg1Δ::rM1r mutant is created through transformation of the strain with a yfg1Δ::rM1r PCR product, an sgRNA gene that targets YFG1, and a CAS9 gene. The M1+ phenotype is selected. For transformation 2, after genotyping, a homozygous yfg1Δ::rM1r mutant is chosen and transformed to create a homozygous yfg2Δ::rM2r mutation. The transformation mix includes a yfg2Δ::rM2r PCR product, an sgRNA gene that targets YFG2, and a CAS9 gene. In addition, in order to eliminate the M1 marker by recombination between flanking repeats, an sgRNA gene that targets M1 itself is also included. For this transformation, the M2+ phenotype is selected. Among M2+ transformants, some are M1−. The M1− transformants are genotyped to identify homozygous yfg2Δ::rM2r mutants. In addition, PCR genotyping is used to verify that yfg1Δ::r is homozygous, marked only with a repeat sequence and not with the entire M1 marker. For transformation 3, a strain homozygous for yfg1Δ::r yfg2Δ::rM2r is chosen, and the strain is transformed to create a homozygous yfg3Δ::rM1r mutation. The transformation mix includes a yfg3Δ::rM1r PCR product, an sgRNA gene that targets YFG3, and a CAS9 gene. In addition, in order to eliminate the M2 marker by recombination between flanking repeats, an sgRNA gene that targets M2 itself is also included. For this transformation, the M1+ phenotype is selected once again, just as it was in the initial transformation. Among M1+ transformants, some are M2−. The M2− transformants are genotyped to identify homozygous yfg3Δ::rM1r mutants. In addition, PCR genotyping is used to verify that yfg2Δ::r is homozygous, marked only with a repeat sequence and not with the entire M2 marker. Manning Y. Huang, and Aaron P. Mitchell mSphere 2017; doi:10.1128/mSphere.00050-17