Paavs1 pdi crisprn

The plasmid for inducible PE2 expression used in this study was derived from pAAVS1-NDi-CRISPRi (Addgene, 73497) by replacing the sequences encoding KRAB-dCas9-mCherry with that encoding PE2 (amplified from pCMV-PE2; Addgene, 132775). CBEs and ABEs were amplified form pCMV_AncBE4max (Addgene, 112094) and ABE8e (Addgene, 138489) respectively and cloned into pAAVS1-PDi-CRISPRn (Addgene, 75300) by replacing with the sequence encoding Cas9. IRES2 EGFP was introduced downstream of AncBE4max and T2A mCherry was fused with ABE8e to monitor transgene expression. To generate iPE2, iCas9, iCBE and iABE cell lines, two million H9 hESCs were co-electroporated with the appropriate knockin vector (5 μg) and plasmids encoding AAVS1-targeting TALENs (2 μg; addgene, 59025 and 59026) using an Amaxa 4D Nucleofector system (Lonza). Serial cell dilutions were then seeded in six-well plates in E8 supplemented with Y-27632 (10 μM). After selection with the appropriate antibiotic, clones were picked, expanded, and screened by treating with dox and staining for Cas9. For genotyping, genomic DNA was extracted with a DNeasy Blood & Tissue Kit. KOD -Multi & Epi (Toyobo, KME-101) was used for junction PCR according to the manufacturer's protocol.

HPSI0214i_kucg-2 cells were engineered to express KRAB-dCas9 from a doxycycline-inducible promoter at the AAVS1 locus^{71 (link)} using pAAVS1-PDi-CRISPRn (a gift from B. Conklin; Addgene, plasmid 73500; http://n2t.net/addgene:73500; RRID: Addgene_73500). The cultures were selected with 100 µg ml⁻¹ G418 until stable colonies originating from single cells formed. The colonies were picked and screened for heterozygous insertion by PCR using two primers flanking the AAVS1 locus (5′-CGAGAGCTCAGCTAGTCTTC-3′ and 5′-CTCTCCCTCCCAGGATCC-3′) and an additional primer binding the insert (5′-GTTCATTCAGGGCACCGGAC-3′). KRAB-dCas9 expression in positive clones was assessed by flow cytometry and immunoblotting after the addition of 2 µM doxycycline. Genome integrity was verified by G-band analysis of expanded clones.