Phelper plasmid

A plasmid for AAV vectors with the human synapsin 1 gene promoter (hSyn) was used for the expression of eGFP alone, or eGFP and hRab35 WT, hRab35 T72A, or hRab35 T72D via P2A peptide-mediated co-expression system. AAV vectors were produced according to the protocol provided by the Salk Institute viral vector core facility (http://vectorcore.salk.edu/index.php) with slight modifications. In brief, HEK-293 cells were co-transfected with a mixture of three plasmids, one AAV plasmid, the pHelper plasmid (Agilent Technologies, Santa Clara, CA, USA), and the serotype 1 plasmid (Vector Core of the University of Pennsylvania), using calcium phosphate. At 72 h after transfection, cells were harvested and lysed by sonication. Then, AAV vectors were collected from the cell lysate by gradient ultracentrifugation using a Beckman NVT90 rotor at 183000 g for 47 min, dialyzed in PBS with D-sorbitol, concentrated by centrifugal filter devices (Millipore, Billerica, MA, USA), and stored at -80 °C. The AAV titer was estimated by quantitative PCR of DNase-I-treated AAV (2-4 × 10¹³ vector genome copies/ml).

AAV293 cells (Agilent Technologies) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Mediatech) supplemented with 10% fetal bovine serum and 1x antibiotic-antimycotic solution (Mediatech). Cells were transfected with three different plasmids (20 µg/plate, each) including AAV transgene plasmids, pHelper plasmid (Agilent Technologies) and AAV2/9 plasmid. Cells were harvested forty-eight hours after transfection. Viral particles in cell lysates were purified using a discontinued iodixanal gradient (Sigma-Aldrich). To determine the viral titer or genome copy number, viral stock was incubated in a solution containing 50 units/mL Benzonase, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, and 10 mM CaCl₂ at 37 °C for 30 minutes. Viral DNA was cleaned with mini DNeasy Kit (Qiagen) and eluted with 40 µL of water. Viral genome copy was determined by quantitative PCR.

(i) AAV2 plasmids. Plasmids pAV2Rep, pCMVAV2Cap, pAAVRep2Cap2 (pR2C2), and pAV2F5tg83luc-CMVmCherry (4.6-kb) have been described previously (35 (link), 76 (link)). AAV2 infectious clone SSV9 (pSub201[-]), containing a full-length AAV2 genome (77 (link)), was a gift from Dr. R. J. Samulski at the University of North Carolina, Chapel Hill (78 (link)).
(ii) Ad plasmids. pAd5E1 cloned with the first 4,031 bp of the Ad5 genome (containing E1 gene) was purchased from OD260 Inc., Boise, ID (#QP-04/pAd1127). pHelper plasmid was purchased from Agilent Technologies, Inc., Santa Clara, CA (#240071).
(iii) pLKO-shRNA constructs. The shRNA-expressing constructs, pLKO-shScram, pLKO-shATM, pLKO-shATR, and pLKO-shDNA-PKcs have been previously described (31 (link)).
(iv) pLentiCRISPRv2 constructs. Three sgRNAs targeting Pol η were cloned in 3 plentiCRISPRv2-Pol η KO vectors (Pol η-1: 5′-GCA CAA GTT CGT GAG TCC CG-3′; Pol η-2: 5′-GAC TGA CCC ATG TGA AAC CA-3′; Pol η-3: 5′-CTG CTC CCA CGG TGA GCT GC-3′). The 3 sgRNAs targeting Pol κ in lentiCRISPRv2-Pol κ KO are Pol κ-1: 5′-TAG GTT CAA CAC ACC TGA CG-3′, Pol κ-2: 5′-ATC CAT GTC AAT GTG CAC TA-3′, and Pol κ-3: 5′-CTT CTC CTT TGT GCT ATC CA-3′. The lentiCRISPRv2-SsgRNA expresses a scramble sgRNA (SsgRNA), containing a random sequence of 5′-GTA TTA CTG ATA TTG GTG GG-3′.