Compozr targeted integration kit aavs1

The purified plasmid library was transfected into K562 cells and genomically integrated using the Zinc Finger Nuclease (ZFN) system for site-specific integration and the CompoZr® Targeted Integration Kit - AAVS1 (SIGMA). Transfections were carried out using Amaxa® Cell Line Nucleofector® Kit V (LONZA). To ensure library representation we performed 10 nucleofections of the purified plasmid library. For each nucleofection, 4 × 10⁶ cells were centrifuged and washed twice with 20 ml of Hank’s balanced salt solution (HBSS, SIGMA). Cells were resuspended in 100 μl solution (warmed to room temperature) composed of 82 μl solution V and 19 μl supplement (Amaxa® Cell Line Nucleofector® Kit V). Next, the cells were mixed with 2.75 μg of donor plasmid and 0.6 μg ZFN mRNA (prepared in-house) just prior to transfection. Nucleofection was carried out using program T-16 on the Nucleofector^TM device, immediately mixed with ~0.5 ml of pre-cultured growth medium and transferred to a 6-well plate with additional 1.5 ml of pre-cultured growth medium. A purified plasmid library was also transfected without the addition of ZFN and served as a control to determine when cells lost non-integrated plasmids.

The methods used for construction of the targeting vector pAAVS1‐EPG and generation of hiPSC‐NSCs are described in the Supplemental material and Figure S1. For transfection, hiPSC‐NSCs at passage 14 (p14) were treated overnight with 10 μmol/L ROCK inhibitor (Y‐27632, Selleckchem) and then dissociated with 0.05% trypsin‐EDTA (Life Technologies). Dissociated hiPSC‐NSCs (1 × 10⁶) were re‐suspended in the R buffer from the Neon Transfection System (Life Technologies) together with 8 μg of the pAAVS1‐EPG vector and 250 ng of mRNAs encoding ZFNs that target the ZFN cleavage site in the AAVS1 locus and were included in the CompoZr^® Targeted Integration Kit‐AAVS1 (Sigma‐Aldrich). Transfection was performed with the Neon Transfection System by using two 20 ms pulses at 1400 V. Transfected cells were plated onto a poly‐l‐ornithine/laminin‐coated (both from Sigma) 6‐well plate. Selection with 2 μg/mL puromycin began at day 7 after transfection. After 10 days, antibiotic‐resistant cells were expanded and aliquots were cryopreserved for further studies. The procedures used for generation of single cell clones and identification of mono‐ and bi‐allelic ZFN‐NSC lines are described in the Supplemental material, Figure S1, and Tables S1 and S2.

The purified plasmid libraries were transfected into K562 cells and genomically integrated using the ZFN system for site-specific integration and the CompoZr® Targeted Integration Kit—AAVS1 (SIGMA). Transfections were carried out using Amaxa® Cell Line Nucleofector® Kit V (LONZA). To ensure library representation, we performed 15 nucleofections of the purified plasmid library for each rationally designed library (frame −1 and frame +1) and five for each native library. For each nucleofection, 4 × 10⁶ cells were centrifuged and washed twice with 20 ml of Hank’s Balanced Salt Solution (SIGMA). Cells were resuspended in 100 μl solution (warmed to room temperature) composed of 82 μl solution V and 19 μl supplement (Amaxa® Cell Line Nucleofector® Kit V). Next, the cells were mixed with 2.75 μg of donor plasmid and 0.6 μg ZFN mRNA (prepared in-house) just prior to transfection. Nucleofection was carried out using program T-16 on the NucleofectorTM device, immediately mixed with ~0.5 ml of precultured growth medium and transferred to a six well plate with additional 1.5 ml of precultured growth medium. A purified plasmid library was also transfected without the addition of ZFN and served as a control to determine when cells lost nonintegrated plasmids.