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. Author manuscript; available in PMC: 2020 Jul 2.
Published in final edited form as: Gene Ther. 2020 Jan 2;27(5):226–236. doi: 10.1038/s41434-019-0120-5

Fig. 1. Construct design and gRNA validation by Surveyor assay.

Fig. 1.

(A) Sequence of AAV vectors represented in cartoon. TBG: thyroxine-binding globulin; ITR: inverted terminal repeats; SA: splicing acceptor; PA: polyA; ITR: inverted terminal repeat; HA: homology arm; HEXM: cDNA of the μ subunit; RE: restriction enzyme site; U6: U6 promoter. (B) Surveyor assay for gRNA activity in MEF cells. Each gRNA construct was transfected into MEF cells, and subsequently harvested for PCR amplification. In order to determine the gRNA cleavage activity of the gRNA constructs, an in vitro Surveyor assay was performed on the PCR products. (C) MUG activities in the liver increased significantly 2 days after hydrodynamic injection of AAV-SaCas9 and AAV-HEXB-gRNA plasmids into Sandhoff mice (n=3). * p<0.05 when comparing treated Sandhoff mice to untreated Sandhoff mice.