Processing
The CRISPR-Cas9 System has been adapted for genome editing in eukaryotic cells. In cultured cells, this method involves adding 3 main components: a CRISPR RNA (crRNA), which contains the DNA-targeting protospacer sequence; trans-activating CRISPR RNA (tracrRNA), which binds Cas9 nuclease; and Cas9 nuclease, which creates blunt-ended cuts in genomic DNA that are repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways.
Figure 1. Enrichment of sorted cells leads to higher editing efficiencies. HEK-293 and Jurkat cells were transfected by electroporation (Neon® electroporation system, Thermo Fisher) with 0.5 µM ribonucleoprotein (RNP: Alt-R® S.p. Cas9 Nuclease 3NLS complexed with Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550, all provided by IDT) and carrier DNA (Alt-R Cas9 Electroporation Enhancer, IDT). Cells subjected to RNP, but without electroporation, provided the background controls and were used to set the gates during fluorescent-activated cell sorting (FACS). Cells were sorted 24 hr post-electroporation, and positive cells were re-plated and grown for an additional 48 hr. A population of the cells was not sorted, but simply re-plated, to serve as the unsorted control. Genomic DNA was isolated using QuickExtract™ solution (Epicentre) after cell incubation for 72 hr. Total editing efficiency was measured using the Alt-R Genome Editing Detection Kit (a T7 endonuclease I assay, IDT).
The Alt-R® CRISPR-Cas9 tracrRNA has previously been optimized by adjusting its length to increase editing efficiency and by adding chemical modifications to increase resistance to nucleases. IDT scientists have conducted additional experiments to determine the best fluorescent dye and dye positions within the CRISPR RNAs for labeling. Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550 is the result of these studies. The addition of a fluorescent dye to the tracrRNA does not affect ribonucleoprotein delivery or genome editing performance. Instead, the fluorescent dye makes it possible to:
- Monitor transfection during optimization of transfection conditions
- Monitor transfection when troubleshooting experiments
- Simplify screening of cells with CRISPR editing events using fluorescent-activated cell sorting (FACS) analysis of transfected cells (Figure 1)
For guidance in using Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550, view the associated application note.
