Cas9 stable cell lines

Figure 1. Structure of stably integrated CRISPR Cas9 nuclease construct. The CRISPR Cas9 nuclease gene was inserted into the GeneCopoeia human AAVS1 Safe Harbor donor clone. This construct was used to knock in the CRISPR Cas9 nuclease into the human AAVS1 Safe Harbor site on chromosome 19 using GeneCopoeia Genome TALER™ TALEN technology. Puromycin (Puro) selection was used for isolation of single clones. CBh: Hybrid CBA promoter. 3X FLAG: N-terminal 3X FLAG tag. NLS: nuclear localization signal. bGH pA: Bovine growth hormone polyadenylation signal. EF1a: Elongation factor 1a promoter. copGFP: Copepod green fluorescent protein. T2A: Element to allow translation of copGFP and Puro independently from the same transcript. SV40 pA: Simian virus 40 polyadenylation signal.

Stable integration at the Human AAVS1 Safe Harbor site ensures consistent, stable expression of the CRISPR-Cas9 nuclease. Cas9 expression from this locus exhibits high activity, as shown by the T7 Endonuclease I mismatch cleavage assay (Figure 2).

Figure 2. Demonstration of stable Cas9 activity in a cell line. Human H1299 cells stably expressing Cas9 nuclease from the AAVS1 Safe Harbor site were transfected with a plasmid carrying an sgRNA for the HUWE I gene. 24 hours post transfection, cells were harvested and used for genomic DNA extraction. Using the GeneCopoeia IndelCheck™ kit, PCR products flanking the sgRNA target site were generated, denatured, reannealed, and treated with (+) or without (-) T7 Endonuclease I, which cleaves mismatched DNA. The presence of two bands below the uncut band at the correct sizes indicates that Cas9 is active in these cells.