Chapter 4 —— 73 —— HRL.S1GS1, AAV-HRC5GC5 and AAV-HRDMDGIN4 (i.e. AK35_pAAVHRL.S1GS1, BF12_pAAV-HRC5GC5 and Y37_pAAV-HRDMDGIN4, respectively); and for the assembly of scAAV donor vectors pscAAV-HRS1 and pscAAV-HMEJS1 (i.e. BG12_pscAAV-HRS1 and BG11_pscAAVHMEJS1, respectively), are available in the Supplementary information. The latter plasmids were generated on the basis of the scAAV construct pscAAV-GFP (Addgene plasmid #32396) [21]. The construct AAVHRLMNAGEX1 contains the gRNA unit GEX1 for site-specific DNA cleavage at LMNA and a matched HR template for LMNA tagging with a mScarlet-I reporter at the N-terminus. The HR template was retrieved from plasmid LMNA_mScarlet-I_Donor (Addgene plasmid #178092) [22]. Finally, the plasmid AT51_pDG6.RSV.DsRed.SV40pA, used as the AAV packaging plasmid, expresses AAV serotype-2 rep and AAV serotype-6 cap proteins together with adenovirus helper functions for AAV production [23]. AdVP vector production, purification and characterization The generation, purification, and characterization of the second-generation AdVs encoding the AAVS1-targeting gRNA GS1 and the regular S. pyogenes Cas9 endonuclease have been detailed elsewhere [24]. The generation of the high-capacity AdVP.C9KARA and AdVP.eC94NLSGS1 particles was done based on the molecular clones U67_pHC-AdVP.C9KARA (Supplementary information) and S90_pHC-AdVP.eC94NLSGS1 (Supplementary information), respectively, using procedures previously described [20,25,26]. In brief, the initial packaging of AdVP genomes into vector capsids was done by transfecting Cre recombinase-expressing and adenovirus type-5 E1complementing PEC3.30 producer cells [20,25,26] seeded at a density of ∼1.6 × 106 cells per well of six-well plates (Greiner Bio-One). The next day, each AdVP molecular clone was first digested with MssI (6.25 μg per clone), to remove the plasmid backbone, and then diluted in 200 μl of a 150 mM NaCl solution. Subsequently, 20.6 μl of a 25-kDa linear polyethylenimine (PEI; Polysciences) solution at a concentration of 1 mg ml-1 was added to the DNA and, after an immediate circa 10-s homogenization in a vortex, was
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