A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad

The landing pad cell line was generated by CRISPR/Cas9-mediated integration of a vector with homology to the hAAVS1 locus. This vector encoded a multicistronic cassette with Hygromycin resistance and nuclear-localized mRuby2 surrounded by a LoxP site and two FRT sites. The BxBI attP site is located between the promoter and the first gene of this cassette (Fig. 4a ). We chose to integrate the landing pad in the hAAVS1 locus due to its low impact on gene expression 16 . We verified the correct integration of the landing pad cassette by PCR in 8 clones ( Supplementary Fig. 3 ) and continued its characterization in clone #8 due to the monoallelic presence of the landing pad. We further confirmed the presence of the landing pad in the cell line by mRuby2 expression (Fig. 4b) .