Fig. 2

Donor plasmid design for matched end joining and HDR-based strategies. A AAVS1 locus is targeted by CRISPR–Cas9 using AAVS1-A1 guide. Cas9-targeted cleavage will create DNA DSBs where the transgene of interest is intended to be inserted. The left and right arms either side of the DSB may be incorporated into the donor template to direct on-target integration (see C and D). B–D The donor plasmids contain a splice acceptor—driven GFP transgene coupled with the puromycin resistance gene, and BFP driven by a CAG promoter on the plasmid backbone. B For the matched end joining strategy, the donor plasmid contains a copy of the AAVS1 sgRNA target site at both flanks of the transgene. Cas9 cleavage in vivo using the AAVS1-A1 guide would cleave the endogenous locus while also generating two DNA fragments from the plasmid, the transgene and the backbone. C, D For homology-directed repair strategies, the transgene is flanked by sequences corresponding to the left and right arms of the endogenous locus. However, the donor plasmids have a mutated PAM sequence of the AAVS1-A1 sequence target, preventing Cas9 cleavage. This ensures the HR-based plasmids remain circular to initiate the HR mechanism. Homology arms are 795 bp and 50 bp in the long and short HA donor plasmids, respectively (C, D). Orange lines indicate left arm homology. Blue lines indicate right arm homology