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. 2018 Feb 9;18(3):foy012. doi: 10.1093/femsyr/foy012

Figure 2.

Figure 2.

Efficient gRNA targeting in K. lactis CBS 2359 enables marker-free gene deletion. (A) Schematic representation of ADE2 editing upon transformation of CBS 2359 with pUDP025 (gRNAKlADE2) and a repair DNA fragment. The primers for diagnostic PCR of transformants are indicated. (B) Colony morphology of CBS 2359 upon transformation with pUDP025 and a marker-free 962 bp repair fragment. (C) Diagnosis of 13 randomly picked red Ade− transformants of CBS 2359 upon transformation with pUDP025 and a 962-bp marker-free repair fragment. Four transformants (HR mutants 1–4) showed a PCR product of 1177 bp corresponding to the deleted allele. The control labeled CBS 2359 and nine transformants (NHEJ mutants 5–13) showed a PCR product of 2838 bp corresponding to the wild-type allele. (D) Sanger sequencing results of purified PCR fragments from nine Ade− mutants (corresponding to mutants 5–13 in panel C) derived from the transformation of CBS 2359 with pUDP025 and repair fragment.