Genome editing: How stable is my CRISPR RNA:Cas9 RNP complex?

Deliver CRISPR RNPs for improved genome editing

Scientists at IDT have demonstrated that for genome editing, CRISPR RNAs and Cas9 protein are most effectively delivered to transfected cell lines as a ribonucleoprotein (RNP) complex [1]. You can obtain both CRISPR crRNAs and tracrRNAs, modified to further enhance genome editing, and S. p. Cas9 3NLS Nuclease from IDT as part of its Alt-R^® CRISPR-Cas9 System.

Store RNPs for future use—get flexibility and continuity

Data from IDT scientists has also confirmed that you can safely complex Alt-R CRISPR-Cas9 System CRISPR RNAs with Alt-R Cas9 Nuclease 3NLS in advance of your experiments, and store these RNPs for future use. This provides researchers with several advantages. The ability to store reagents that retain full activity means you avoid discarding unused material and get the most data from your investment. Use of the same reagents maintains consistency across subsequent experiments. And, preparing reagents in advance saves valuable time during experiments, allowing you to focus on other critical steps in the protocol.

Stored CRISPR RNPs provide the same genome editing efficiency as freshly made RNPs

Figure 1 shows data from RNPs stored for 10 weeks prior to use in a genome editing experiment). RNPs have no loss in activity when stored for 10 weeks at –80°C or 4°C diluted in any of the tested buffers. RNPs stored at –20°C showed slightly reduced genome editing activity for one of the HPRT sites targeted (Site 2).

This experiment also illustrates that site selection can affect genome editing efficiency, underlining the importance of testing 2–3 target sites. In this case, genome editing was consistently more efficient at HPRT Site 1 (38087) than HPRT Site 2 (38285).

IDT scientists have also noted that the S. p. Cas9 Nuclease 3NLS itself is robust. There was no loss in genome editing activity when this Cas9 nuclease alone was diluted to 1 µM and stored at 4°C or –80°C for 7 weeks, though again, a slight loss in activity was seen for diluted enzymes stored at –20°C (data not shown). Thus, the enzyme can be diluted to a working concentration and reused for later experiments with no loss in activity. Likewise, enzymes at stock concentration or diluted to 1 µM and unintentionally left out on the bench overnight or stored in a freezer that has lost temperature, is still effective.

Figure 1. CRISPR RNA:Cas9 RNPs stored for 10 weeks at –80°C or 4°C provide the same high level of genome editing as freshly complexed RNPs. CRISPR RNAs (Alt-R CRISPR-Cas9 System, IDT) targeting each of 2 HPRT gene sites (Site 1 = 38087, Site 2 = 38285) were complexed with S. p. Cas9 3NLS Nuclease (Alt-R CRISPR-Cas9 System, IDT) as an RNP in each of 3 buffers (Cas9 Buffer, Opti-MEM media, PBS). The RNP complexes were stored at 4°C, –20°C, and –80°C for 10 weeks and then reverse transfected into HEK293 cells (RNAiMAX Transfection Agent, Thermo Fisher Scientific). 3 biological replicates were included. Freshly complexed RNPs diluted to 1 µM in Cas9 Buffer were also transfected as a control. Genomic DNA, isolated 48 hr post-transfection, was subjected to a T7EI mismatch endonuclease assay to evaluate genome editing efficiency. Genome editing efficiency was just as high with RNPs stored for 10 wk at –80°C or 4°C as with freshly complexed RNPs.

 

Recommendations for storing CRISPR RNPs

The data presented here confirms that you can safely complex Alt-R CRISPR-Cas9 System CRISPR RNAs with S. p. Cas9 Nuclease 3NLS in advance of your experiments, thus providing reagent consistency, saving laboratory time and reagents, and giving you more schedule flexibility. RNP complexes stored in this way provide the same high level of genome editing as freshly complexed RNPs.

We recommend that you store CRISPR RNPs at 4°C for no more than 2 weeks, as bacterial or fungal growth may occur as a result of accidental contamination. If a longer storage duration is needed, we recommend storing your RNPS at –80°C.