MISSION™ Ribonucleoprotein (RNP) Protocols

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

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. 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

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.

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.

General Recommendations

  • Preparation of cells—Approximately 18–24 hours before use, plate cells in complete growth medium. For most cell types, cultures should be 50–80% confluent at the time of transfection.
  • SygRNA® Resuspension—Resuspend dried SygRNA® sgRNAs or crRNAs and tracrRNAs to a concentration of 20 µM (20 picomoles per µl) each in 10 mM Tris-containing buffer of pH between 7 and 8.
  • Cas9 Protein Resuspension—Resuspend the lyophilized protein with the supplied reconstitution solution (Product number RSOLUTION).  For 250 µg vials, add 50 µL of reconstitution solution to achieve a concentration of approximately 5 mg/mL. For 50 µg vials,  add 30 µL of Reconstitution Solution to achieve a concentration of approximately 1.7 mg/mL.
  • Assembly of RNP—Assemble SygRNA®:Cas9 Protein complexes (RNP) on ice, immediately before use. It is recommended to prepare RNP in a molar ratio between 1:1 to 5:1 (guide RNA:Cas9 protein). Further optimization may be required.  The guide RNA can be synthetic or in vitro transcribed (IVT). We provide custom SygRNA® synthetic single guide RNAs (sgRNA) or synthetic crRNAs and tracrRNAs. When using synthetic crRNA and tracrRNA, the two RNA molecules should be used in equal molar amounts. Annealing of the crRNA and tracrRNA is optional.


    RNP complex

  • Genomic DNA harvest and mutation detection—Allow the cells to grow 48–72 hours post transfection before harvesting genomic DNA. There are many methods to detect indels produced by CRISPR systems. The most commonly used methods include NGS based sequence analysis, mismatch detection assays, and Sanger based sequencing analysis.

Related Products

Table 1: MISSION™ Cas9 Protein Products

 

Product No. Description
CAS9GFPPRO Cas9-GFP Protein from Streptococcus pyogenes, fused with enhanced GFP, recombinant, expressed in E. coli, 3X NLS 
ECAS9GFPPR eSpCas9-GFP Protein from Streptococcus pyogenes with mutations conferring enhanced specificity, fused with enhanced GFP, recombinant, expressed in E. coli, 3X NLS 
CAS9PROT Cas9 Protein from Streptococcus pyogenes, recombinant, expressed in E. coli, 1X NLS
ESPCAS9PRO eSpCas9 Protein from Streptococcus pyogenes with mutations conferring enhanced specificity, recombinant, expressed in E. coli, 1X NLS
DCAS9PROT dCas9-3XFLAG™-Biotin Protein from Streptococcus pyogenes with D10A and H840A mutations, recombinant, expressed in E. coli, 1X NLS
CAS9D10APR Cas9-D10A Nickase Protein from Streptococcus pyogenes with D10A mutation, recombinant, expressed in E. coli, 1X NLS
FNCAS9PROT   FnCas9 Protein from Francisella novicida, recombinant, expressed in E. coli, 1X NLS

 


Table 2: Other Related Products
 

Product No. Description
Custom Guide RNA Order custom guide RNAs on line in various formats
TRACRRNA05N SygRNA Cas9 Synthetic tracrRNA
TRACRRNAMOD SygRNA Cas9 Synthetic modified tracrRNA
T1706 TransIT®-CRISPR
XTG360-RO X-tremeGENETM 360 Transfection Reagent
FNCAS9TRACR-5NMOL SygRNA® FnCas9 tracrRNA
Embryo Culture Media Order various types of mouse embryo culture media and mouse embryo tested reagents
Cell Culture A complete catalog of cells and cell culture reagents

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).