CRISPR Cas9/gRNA ribonucleoproteins (RNP) are composed of a CRISPR RNA (crRNA) and tracrRNA duplex complexed with a Cas9 protein. Unlike traditional Cas9/gRNA constructs, the crRNA and tracrRNA oligonucleotides are delivered as intact complexes, overcoming the need for the cell's own transcription machinery to express the CRISPR components. As a result, Cas9 RNPs have multiple advantages over other systems:
- Cas9 RNPs edit at high efficiency quickly after delivery
- Cas9 RNPs clear quickly from cells via protein degradation
- Off-target cleavage is reduced compared to CRISPR plasmids
- Cas9 RNPs are ideal for in vivo gene editing applications
GenScript is pleased to offer a Cas9 RNP design service featuring pre-designed and synthesized crRNA and tracrRNA oligonucleotides, that are ready for Cas9 complexing and packaging for easy gene editing.
Advantages of GenScript's Cas9 RNP service:
- gRNA design included: choose from Broad-validated gRNA sequences or design your own
- Delivered as duplexed crRNA: tracrRNA oligos no need for in vitro transcription
- Ready for transfection: just prepare the complex and deliver into your cells!
- Flexible quantity options
Cas9 RNP Service features
The Cas9 RNP service includes three components: (1) a customized crRNA, (2) a conserved tracrRNA and (3) Cas9 protein. The crRNA consists of a 20 nt sequence that is complementary to an endogenous target gene. When complexed with tracrRNA and Cas9 protein, a double strand break will be created at a locus complementary to the 20 nt guide RNA sequence, 3-4 bp upstream from a PAM sequence (5'-NGG-3'). Since crRNA sequence design utilizes standard gRNA design principles, sequences can be easily selected from our gRNA database, or designed de novo using our gRNA design tool. The synthetic crRNA:tracrRNA will be delivered pre-duplexed and ready for complexing with Cas9 protein.
|Catalog #||Product Name||Quantity||Turnaround Time||Price|
GenCRISPR Cas9-2NLS nuclease
10 μg (1 mg/ml)*
HPRT positive control
*10 μg cas9-2NLS nuclease equal to 62.5pmol
Cas9 RNP workflow
Figure 1: Cas9 RNP workflow
Due to the simplicity of the Cas9 RNP system, there are only two steps required for using synthetic crRNA:tracrRNA oligos: (1) incubate the oligo with Cas9 protein to form the RNP and (2) deliver the RNP into the cells (Figure 1). An HDR template can also be co-delivered with the Cas9 RNP for gene knock-in. Prior to experimentation, we recommend first testing your Cas9/gRNA complex in vitro and in test cell lines to ensure cutting efficiency. Details on how to form the RNP complex and perform the preliminary tests are included in the user manual.
Delivery of the Cas9 RNP complex into cells is typically performed by electroporation or via lipid-mediated transfection. Selecting the best delivery method for your cell type may require some experimentation; however, the method that works best for Cas9/gRNA plasmids may also work well for RNP. More details on this process can also be found on Addgene's website.
- Synthetic crRNA:tracrRNA complexes are delivered in 2 nmol, 5 nmol, 10 nmol or 20 nmol quantities
- Available in lyophilized format
- Cas9 nuclease reaction buffer is included with the Cas9 protein (10 μg or 25 μg)
- All RNP products are delivered with a user manual
How to store and use crRNA:tracrRNA oligonucleotides? Read More »
Keep the crRNA:tracrRNA oligonucleotides tightly sealed at -20℃ prior to use and avoid repeated freeze-thaw cycles. We recommend working in a sterile environment, using RNAse-free pipette tips and tubes. To dissolve the RNAs, first centrifuge at 6000g for 10 seconds at 4℃, then add DEPC water. Use the following table to dissolve the crRNA:tracrRNA oligonucleotides to 50 pmol/μl.
|Normalized amount delivered (nmol)||DEPC water (μl)|
How to store and use Cas9 protein? Read More »
Keep the Cas9 vial sealed until use and avoid repeated freeze-thaw cycles, as either may reduce the activity of Cas9 protein. Prior to use, dilute the protein solution using a diluent buffer (10 mM Tris, 300 mM NaCl, 0.1 mM EDTA,1 mM DTT, 50% Glycerol to pH7.4 at 25℃).
What amount of Cas9 protein and crRNA:tracrRNA should I use? Read More »
It is recommended to use the same amount of Cas9 protein and crRNA:tracrRNA. We have found that 3pmol of Cas9 protein (240ng) and 3pmol of crRNA:tracrRNA are sufficient for genome editing.
How do I use the HPRT positive control? Read More »
RNP gene editing efficiency testing is best performed in easy-to-handle human cell lines, such as HEK293 cells.
- Seed well-dissociated cells one day (16-24hrs) prior to transfection in media lacking antibiotics.
- Incubate cells with transfection reagents. 30-70% confluency at the time of transfection is recommended, as high confluency can negatively impact efficiency.
- Harvest cells approximately 48hrs after transfection and extract genomic DNA for analysis.
- PCR amplify genomic fragments using GenScript Human HPRT Primers and verify genome editing efficiency via sequencing or T7E1 digestion assay. Verify that the expected PCR product is obtained.
Have questions? GenScript's Ph.D.-level service representatives are available 24 hours a day, Monday through Friday, to assist you.