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

Prepare and store CRISPR RNA:Cas9 RNP complexes for several weeks with no loss in activity

You can safely complex CRISPR RNAs with Cas9 in advance of your experiments and store these RNPs for future use. Store CRISPR RNPs at 4°C for up to 2 weeks, or at –80°C long-term. RNP complexes stored in this way provide the same high level of genome editing as freshly complexed RNPs.

Jul 25, 2016

Revised/updated Feb 3, 2017

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.

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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.

RNP stability testing

RNP stability experiments used crRNA sequences targeting 2 distinct HPRT gene sites. CRISPR RNAs and Cas9 nuclease were complexed following the instructions in the Alt-R CRISPR-Cas9 System User Guide. RNP complexes (1 µM in Cas9 Buffer, Opti-MEM® media, or PBS) were stored at 4°C, –20°C, and –80°C for 48 hours, 2 weeks, and 10 weeks. RNPs were then transfected into HEK293 cells using RNAiMAX Transfection Agent (Thermo Fisher Scientific) following the RNP lipofection protocol described in in the Alt-R CRISPR-Cas9 System User Guide. The experiment included 3 biological replicates; freshly complexed RNPs diluted to 1 µM in Cas9 Buffer were also transfected as a control.

Transfected cells were plated and grown for 48 hr. Genomic DNA was subsequently isolated and subjected to a T7EI mismatch endonuclease assay, as described in the Alt-R CRISPR-Cas9 System User Guide.

T7EI mismatch endonuclease assays

We currently recommend using T7 endonuclease I (T7EI) for CRISPR mutation detection. The T7EI method for genome editing analysis is simple and provides clean electrophoresis results. T7EI endonuclease is compatible with a broad range of PCR buffers and does not usually require purification of the PCR product prior to digestion. Note that T7EI activity is sensitive to the DNA:enzyme ratio, as well as incubation temperature and time [2]. T7EI is able to recognize insertions and deletions of ≥2 bases that are generated by NHEJ activity in CRISPR experiments [3]. Note that because T7EI does not recognize 1 bp indels, T7EI actually underrepresents the total editing.

References

  1. Packer H. (2016) Improve your genome editing with the Alt-R S.p. Cas9 Nuclease 3NLS and modified crRNAs. [Online] Coralville, Integrated DNA Technologies.
  2. Mean RJ, Pierides A, et al. (2004) Modification of the enzyme mismatch cleavage method using T7 endonuclease I and silver staining. Biotechniques, 36(5):758–760.
  3. Vouillot L, Thélie A, Pollet N. (2015) Comparison of T7EI and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3: Genes|Genomes|Genetics, 5(3):407–415.

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