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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

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 (see data in the article, Improve your genome editing with the Alt-R® S.p. Cas9 Nuclease 3NLS and modified crRNAs). 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

Here review data produced by IDT scientists confirming 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.

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. Note that the T7El assay underestimates total editing (see the sidebar, T7EI mismatch endonuclease assay for genome editing analysis). Results for storage at 10 weeks are shown in Figure 1.

T7EI mismatch endonuclease assay for genome editing analysis

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 [1]. T7EI is able to recognize insertions and deletions of ≥2 bases that are generated by NHEJ activity in CRISPR experiments [2]. Because T7EI does not recognize 1 bp indels, T7EI underrepresents the total editing. For a protocol, see the Alt-R® CRISPR-Cas9 System User Guide.

References

  1. 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.

  2. 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.

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 ((see sidebar, RNP stability testing,). 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 same high level of genome editing as freshly complexed RNPs

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


We recommend that you store CRISPR RNPs at 4°C for up to 2 weeks (longer storage may result in bacterial or fungal growth in case of introduced contamination), or at –80°C for long-term.

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.

Learn more about using CRISPR RNA:Cas9 RNP complexes for genome editing from the articles cited in the Additional reading section below. The RNP lipofection protocol used for this experiment is described in detail in the Alt-R CRISPR-Cas9 System User Guide.

Product focus—genome editing with Alt-R® CRISPR Reagents

Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
  • Efficient delivery of the RNP with lipofection or electroporation
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.


Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation

Learn more about the Alt-R CRISPR-Cpf1 System.


CRISPR support tools

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.

Additional reading

6 pieces of data that will change how you set up your CRISPR-Cas9 experiments—To improve the efficiency of CRISPR-Cas9 genome editing, IDT scientists evaluated several factors—Cas9 delivery, crRNA and tracrRNA length, which gRNA formats provide the most efficient on-target editing and elicit the least toxicity, the importance of protospacer size and site selection—that influence how we design and perform genome editing experiments. Review the data and results for 6 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

Webinar: Alt-R® CRISPR-Cas9 System ribonucleoprotein delivery optimization—Genome editing using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) provides excellent editing efficiency, while reducing off-target editing and cell death. Watch this recorded presentation to find out how to easily generate and deliver CRISPR RNAs and Cas9 nuclease in an RNP format, using the optimized Alt-R CRISPR-Cas9 System.

Successful CRISPR genome editing in hard-to-transfect cells (i.e., Jurkat cells)—Use the conditions presented here for Clone E6-1 Jurkat cells as a starting point for optimization of CRISPR reagent delivery in cell types requiring electroporation.

Improve your genome editing with the Alt-R® S.p. Cas9 Nuclease 3NLS and modified crRNAs—Product Spotlight: Methods for CRISPR-Cas9 genome editing are diverse, but not all of them perform equally well. IDT now offers the Alt-R™ S.p. Cas9 Nuclease 3NLS that, when used in combination the optimized Alt-R CRISPR crRNA and tracrRNA, provides a highly effective editing solution that is also easy to use.

Author: Ashley Jacobi is a staff scientist, and Ellen Prediger, PhD, is a senior scientific writer, both at IDT.

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