Fig. 2
From: NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger
Integration of an inducible expression cassette at the pyrG locus by HDR and variants of MTR. (A) Promoter region (light grey) and CDS (dark grey) of pyrG with Cas9-mediated double-strand break. PCR3 of the parental strain A. niger pyrGm1 gives an amplicon with 4152 bp. The donor fragment consists of a 5’ flank homologous arm (blue), an inducible cassette for heterologous expression of the phosphoketolase Xfspk, followed by the promoter coxA (orange). The 3’ flanking sequence is a truncated pyrG CDS (dark grey). (B) Integration via HDR was verified by PCR1-1, PCR2, and PCR3-1 with amplicon sizes of 3793, 6841, and 10,692 bp, respectively. (C) Integration via MTR with NHEJ on the 3’ end and HDR at the 5’ end of the double-strand break. In clone 1, NHEJ of sequences form a palindrome. In clone 2, the donor DNA is shortened by end resection prior to the NHEJ event. PCR1-2 and 3 − 2 are 5085 bp and 11,984 bp for variant 1 and 4974 and 11,873 bp for variant 2