Cas9 coding pspcas9 bb 2a puro

Plasmids were electropoated using Nucleofector™ Kits for Human Dermal Fibroblast (Lonza) according to the manufacturer’s protocol. Briefly, after washing with PBS, iPSCs were treated with Accutase (Life Technologies) to dissociate single cells. These cells were suspended with Nucleofector solution and Supplement (Lonza) and mixed with gRNA expression vector, Cas9 coding pSpCas9(BB)-2A-Puro (Addgene) and double-strand plasmid DNA for donor. The donor plasmid was constructed by cloning the flanking regions of rs3851179 amplified by primers as follows; F: 5′- ACCCATCACCCTTCTGTTTG-3′ and R: 5′-TTTTCCAGCAAGTTGGGTTC-3′. Puromycin selections were started at 24 hours after electroporation and continued 3 to 4 days at the concentration of 0.75 µg/mL. After withdrawal of puromycin, cells were cultured in mTeSR until suitably sized colonies appeared. Colonies were picked up and genomic DNAs were extracted using QuickExtract™ DNA Extraction Solution 1.0 (epicentre) according to manufacturer’s protocol. Target site was amplified by PCR using primers as follows; F: 5′- CCCGCTTCATAGGGTTATTG-3′ and R: 5′- AACTCACCCCAGTCTCTTGC-3′. We confirmed suspected mutant clones by direct sequencing of the PCR products. We used Sanger sequencing of iPSCs at rs3851179 to verify independent isogenic lines homozygous for either the G or A variant.