Fig. 3: In vitro design and validation of all-in-one, self-inactivating rAAV:HDR vectors. | Nature Communications

Fig. 3: In vitro design and validation of all-in-one, self-inactivating rAAV:HDR vectors.

From: Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

Fig. 3

a Schematic of four different vector designs of rAAV:HDR constructs containing an sgRNA expression cassette, Nme2Cas9, and a donor DNA (<500 bp) with and without flanking sgRNA target sites. b Agarose gel electrophoresis (1% agarose) of linearized AAV:HDR plasmids. Asterisks indicate self-inactivated AAV:HDR plasmids that were cloned using anti-CRISPR-protein-expressing E. coli DH5α. DNA size marker size is 1 kb. Multiple plasmids were analyzed in this fashion three separate times, with consistent results. c Schematic of conventional E. coli DH5α and the recombined, anti-CRISPR-protein-expressing derivative used to successfully clone the self-targeting rAAV:HDR:cleaved plasmids. d Efficiencies of NHEJ and HDR events depicted as percentages of mCherry- and GFP-positive cells, respectively, obtained after transfection of AAV:Nme2Cas9:sgRNA and dsDNA GFP donor in trans), or of rAAV:HDR constructs in TLR-MCV1 HEK293T cells. The sample size represents independent transfection experiments (n = 2 of Nme2Cas9, sgRNA, and dsDNA donor in trans delivery; while n = 4 biological replicates of in cis delivery of AAV:HDR plasmids Designs A, B, C, and D). e Schematic of modified rAAV packaging transfection system using anti-CRISPR protein plasmid to block self-targeting by Nme2Cas9 expression in packaging cells during production.

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