Cas9 stable cell lines

CRISPR-Cas9 stable cell lines


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.

 
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