Protocols and Considerations for Ribonucleoprotein (RNP)-Based Genome Editing

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system was discovered in bacteria, where it functions as an adaptive immune system against invading viral and plasmid DNA. In this system, short DNA sequences (spacers) from invading viruses are incorporated at CRISPR loci within the bacterial genome and serve as a memory of previous infections. Reinfection triggers complementary mature CRISPR RNA (crRNA) to find a matching viral sequence. Together, the crRNA and trans-activating crRNA (tracrRNA) guide CRISPR-associated (Cas) nuclease to cleave double-strand breaks in the corresponding foreign DNA sequences.1

The type II prokaryotic CRISPR “immune system” has been engineered to function as an RNA-guided genome-editing tool that is easy, and quick to implement. Here, we describe two different recombinant Cas9 proteins: Cas9 (wild-type SpCas9) and enhanced specificity Cas9 (eSpCas9), which has been shown to reduce off-target cleavage.2 These proteins can be combined with SygRNA® synthetic crRNAs and tracrRNAs to form ribonucleoprotein (RNP) complexes that target the specific genomic locus of interest (Figure 1).

Getting Started - Gene Editing with Cas9 Proteins

Three Component CRISPR Cas9 System.
 

Figure 1. Three Component CRISPR Cas9 System. The Cas9 ribonucleoprotein is made up of the Cas9 protein and a guide RNA, which can be divided into a tracrRNA and a crRNA. The crRNA is variable and complementary to the target of interest, while the tracrRNA sequence is static.

Although the CRISPR system can be delivered to cells via plasmids, direct introduction of Cas9 RNP strengthens and expands the applications of CRISPR genome modification technology by eliminating the possibility of plasmid DNA integration into the host genome. This method also reduces the risk for off-target effects due to the rapid degradation of the RNP after delivery (Figure 2). In many cases, Cas9 RNP results in efficient genome modification with a higher specificity when compared to cells transfected with Cas9 plasmid.1,3,4,5 This RNP technology has broad applications and has been shown to function in both mammalian and plant systems.6 Furthermore, Cas9 RNP delivery holds great promise for therapeutic applications including the recent successful generation of knock-in primary Human T cells.7

Figure 2. eSpCas9 reduces off-target cleavage compared with WT SpCas9. In this experiment, K562 cells were nucleofected with WT SpCas9 or eSpCas9 and synthetic tracrRNA and EMX1-targeted crRNA. A CEL-1 assay showed equal cleavage efficiency between WT SpCas9 and eSpCas9, while cleavage at a known off-target site was reduced when eSpCas9 was used compared to Cas9.
 

General Considerations

We recommend using your preferred method to introduce nucleic acids into your cells of interest. We provide a variety of transfection reagents, cell culture media and plates, and custom DNA primers for detection of CRISPR-mediated genome editing. For your reference, we have suggested protocols below.

Recommendations

  • Assemble SygRNA® Reagent:Cas9 Protein complexes (RNP) on ice, immediately before use.
  • In all instances, combine equal molar amounts of crRNA:tracrRNA.
  • For transfection of RNP using a transfection reagent, prepare RNP using a ratio between 1:1:1 to 5:5:1 crRNA:tracrRNA:Cas9 protein.
  • For nucleofection of RNP, prepare RNP using a ratio between 1:1:1 to 5:5:2 crRNA:tracrRNA:Cas9 protein.

In general, the steps required for successful introduction of Cas9 RNP into cultured and primary cells are:

  1. Prepare SygRNA® crRNA and tracrRNA reagents
  2. Prepare cells
  3. Assemble Cas9 RNP
  4. Transfect cells with RNP
  5. Harvest and assay cells

Troubleshooting

If no cutting is observed and there is reason to suspect an experimental flaw is at fault, the following considerations may aid in troubleshooting the experiment.

 
Suspected Issue Solution
The Cas9 protein has denatured after long-term storage in dilution buffer. The provided dilution buffer is only recommended for immediate use. For long-term storage, keep the protein lyophilized or resuspended in the provided Reconstitution solution at -20 ℃.
The Cas9 protein has been thawed and refrozen too many times. The Cas9 protein has been shown to withstand several rounds of freezing and thawing without sacrificing cutting activity, but aliquoting the protein into smaller quantities upon resuspension will allow this potential issue to be avoided.
The crRNAs and tracrRNAs need to be annealed before complexing with the Cas9 protein. Although an annealing step is generally not needed, it has shown to increase cutting in rare cases.8 To anneal the crRNA and tracrRNA, mix them in the desired ratio and incubate the mixture for 5 minutes at 95 ℃, then place the mixture on ice for 20 minutes.
The crRNAs and tracrRNAs are degraded. Under normal cell culture conditions, synthetic RNA modifications are not needed for stabilization; however, for certain cell lines, this may be necessary. Modifications are available through us.
The transfection or nucleofection is not working or is too toxic. For any transfection reagent or nucleofection, optimize the protocol for each cell line used. Refer to the manufacturer’s protocol for further assistance.
In vitro transcribed RNA is low quality or degraded. For optimal performance, use only quality-verified IVT RNA.

 

 References

  1. Kim, S. et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24, 1012–1019 (2014).
  2. Slaymaker, I.M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).
  3. Zuris, J.A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nature Biotechnology 33, 73–80 (2015).
  4. Lin, S. et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife (2014).
  5. Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology 208, 44–53 (2015).
  6. Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications 8 (2017).
  7. Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. PNAS 112, 10437–42 (2015).
  8. Mekler, V. et al. Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3'‑terminal segment of guide RNA. Nucleic Acids Research 44, 2837–2845 (2016).