Fig. 1
From: Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression
Overview on the CRISPR/Cas9 plasmid assembly. A. Scheme of the multiple overlap PCR to generate the 248 bp fragment. This fragment was assembled by PCR using two primers containing the sequence specific to the gRNA target and four generic primers. The obtained product includes a hammerhead ribozyme, the complementary sequence to the target DNA, the sequence for expression of the gRNA, a hepatitis delta virus (HDV) ribozyme for cutting at the 5’ and 3’ ends of the gRNA and BsaI restriction sites on the 3’ and 5’ ends. B. Overview on the two GGAs to generate the final CRISPR/Cas9 plasmid. The initial GGA using BsaI, leads to the ligation of the 248 bp-fragment and the BB1_L_23_syn_BsaI plasmid. The product of this assembly is then assembled with the plasmid bearing the Cas9 expression cassette via BsbI