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

Figure 6.

Figure 6.

Simultaneous deletion of OpADE2 and OpYNR1 alleles using a single ribozyme flanked gRNA array in O. parapolymorpha CBS11895. (A) Representation of the gRNA array expression cassette in pUDP123. The dual gRNA array was under the control of the RNA polymerase II promoter ScTDH3 and ScCYC1 terminator. Each gRNA was flanked on its 5΄ by a hammerhead ribozyme (HH represented in orange) and on its 3΄ by a hepatitis delta virus (HDV represented in bronze) ribozyme which were separated by a 20-bp linker. Upon ribozyme self-cleavage, the mature gRNAs are released. The OpADE2 guiding spacer (in purple), the OpYNR1 guiding spacer (in yellow) and the constant structural gRNA fragment (in green) are indicated. (B) Schematic representation of the OpADE2 and OpYNR1 loci of CBS11895. The primers for the validation of transformants are indicated. (C) Sanger sequencing results of OpADE2 and OpYNR1 editing site of five randomly picked red Ade– mutants that have lost ability to grow on nitrate. Transformant labeled Transf#2 was renamed IMD034.