Fig. 1: Schema for creation of the rAAV-donor. | Communications Biology

Fig. 1: Schema for creation of the rAAV-donor.

From: Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase

Fig. 1

Step 1: left (LHA) and right (RHA) homology arms were created by PCR from human genomic DNA template using PCR primers tailed with restriction enzyme sites (REs; RE 1, 2 for LHA and RE 3, 4 for RHA) for subsequently cloning into the pAAV acceptor. Step 2: The left and right homologous arms were digested and sequentially cloned into the pAAV-SEPT-Acceptor plasmid, which has been built with a Neomycin (NeoR) gene-trap cassette. Step 3: The extra 20 bp from upstream exon (primer 2 sequence) was introduced in the beginning of the RHA of the pAAV-SEPT-Acceptor by site-directed mutagenesis (SDM). Step 4: The desired substitution (K700E mutation) was introduced in the right arm of the pAAV-SEPT-Acceptor by SDM (shown in magenta).

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