Geneart dna string

Cells were transfected with 250 ng U6-gRNA GeneArt DNA String (Thermo) pairs (Supplementary Table 1) and 250 ng pSPCas9(BB)-2A-GFP (PX458) into dLP cells with mAb integrated into LP2. Three days post transfection, cells were sorted for GFP-positive single cells and genomic DNA was assayed by PCR for exon excision (Supplementary Table 2). pSPCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138). 44

Single-and multi-LP cell lines in LP2, LP8 and LP20 loci were constructed by homologous recombination with CRISPR/Cas9 as described 26 with the following changes. Targeted integrations were performed by co-transfecting 500 ng of circular LP donor vector with 40 ng of pSpCas9(BB) (pX330) plasmid and 150 ng of U6-gRNA GeneArt DNA String (Thermo). About 10 5 cells were transfected in triplicate with a Neon electroporator (Invitrogen) using 10 pulses of 1560 V and 5 ms width, and seeded in 24-well plate. Three days post-transfection cells were combined and transferred to a 125 mL flask with 10 mL CD-CHO media, recovered for 1 day, and on day 4 subjected to antibiotic selection with either hygromycin (200 µg/mL) or blasticidin (2-20 µg/mL) for two weeks followed by clonal sorting with FACS. Clonal cells were verified with diagnostic PCR using locus-specific and LP-specific primers (on-target integration) and backbone-specific primers (off-target integration). Clones exhibiting locus-specific and backbone-free integration, and stable and homogenous LP expression were isolated and subsequently used for mAb payload and genetic circuits integrations. pX330-U6-Chimeric_BB-CBh-hSPCas9 was a gift from Feng Zhang (Addgene plasmid # 42230). 43