AAVS1-DOX-KRAB-dCAS9-puro plasmid construction

Puro-Cas9 donor was a gift from Danwei Huangfu (Addgene plasmid # 58409)(³⁸). This plasmid was restriction enzyme digested with AgeI and SalI. The larger band containing the AAVS1 homology arms, tet operator, and puromycin resistance gene were in the larger of the two bands (7184 bp). This band was cut from a 0.7% agarose gel and purified by the Zymo Gel purification kit. pHR-SFFV-KRAB-dCas9-P2A-mCherry was a gift from Jonathan Weissman (Addgene plasmid # 60954) (¹⁷). This plasmid was PCR amplified with primers AT574 and AT575 using KAPA HIFI Mastermix (³⁹). Primer sequences can be found in Table S2. The PCR product was purified using the Qiagen column purification kit. The two pieces were combined using the GeneArt Seamless PLUS Cloning and Assembly Kit at a ratio of 2:1 PCR product: plasmid digest. This generated a novel plasmid containing KRAB-dCAS9 with a P2A followed by mCherry under the tet operator. These were flanked with AAVS1 targeting homology arms as well as a puromycin resistance gene that is inserted in-frame in the AAVS1 gene. Complete plasmid sequencing was performed by Massachusetts General Hospital DNA Core by Next-Gen sequencing and automated sequence assembly. The clone used had a mutation in mCherry gene that resulted in a reduction in signal allowing for the utilization of mCherry as a selection marker for pUCM-CLYBL-Ngn1/2 cassette integration sequentially after integration of the DOX-KRAB-dCAS9-puro cassette.