Synthetic crRNA/tracrRNA

CRISPR/Cas9 ribonucleoprotein (RNP) complexes are composed of a CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA) duplex and Cas9 protein. The crRNA consists of a 20 nt sequence that is complementary to a genomic target. crRNA sequence design utilizes standard gRNA design principles and sequences can be easily selected from the GenScript gRNA database or customized using the GenScript gRNA design tool. 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 protospacer adjacent motif (PAM) sequence (5'-NGG-3'). Unlike traditional CRISPR Plasmids, CRISPR/Cas9 ribonucleoproteins are delivered as intact complexes, and do not require cellular expression.

CRISPR RNA/Cas9 Protein Workflow

Only three steps are required for using synthetic CRISPR RNA oligos:

1. Incubate the crRNA and tracrRNA with the annealing buffer
2. Incubate the duplexed crRNA:tracrRNA with Cas9 protein to form the active CRISPR/Cas9 ribonucleoprotein. Optionally, an HDR (homology dependent repair) template can also be co-delivered with the Cas9 RNP for targeted knock-in.
3. Deliver the ribonucleoprotein mix into the cells

Prior to experimentation, we recommend first testing your CRISPR/Cas9 complex in vitro and in test cell lines to ensure cleavage and transfection efficiency. Delivery of the CRISPR/Cas9 complex into cells is typically performed by electroporation or via lipid-mediated transfection.

CRISPR Handbooks and Protocols

CRISPR Handbook

CRISPR/Cas9 Protein & RNA FAQs

• What are your CRISPR plasmid delivery specifications? Read More »

Deliverables:

• 2-20 nmol of crRNA and/or tracrRNA
• Available in lyophilized format

QC:

• HPLC verification
• Quality assurance certificate

• What are your Cas9 Protein delivery specifications? Read More »

Deliverables:

• 10-25 μg of Cas9-2NLS protein
• Available in lyophilized format

QC:

• Quality assurance certificate

• How many crRNA sequences are needed for targeted knock-out? Read More »

A minimum of 3 crRNA sequences are recommended to ensure knock-out and experimental accuracy. Independently obtained knock-out mutants provide redundancy to safeguard against any hidden off-target effects.

• Do I need an all-in-one or dual vector system? Read More »

All-in-one vector systems have two main advantages:

• Cells only need to be transfected once.
• gRNA/Cas9 expression is driven in an ideal 1:1 ratio.

Dual vectors, where Cas9 and gRNA are expressed independently on separate constructs, are more suitable if you plan to express multiple gRNAs for multiplex targeting.  For these applications, Cas9 should first be stably expressed in the cell line, after which the cells can be transfected with different gRNA vectors to generate a cell pool.

• How to store and use crRNA:tracrRNA oligonucleotides? Read More »

Keep the crRNA and 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.

For 5X Annealing buffer:

• 1. Centrifuge tubes before opening to ensure RNA oligos are at the bottom of the tube.
• 2. Resuspend both oligos in Nuclease-Free water to reach the appropriate final concentration, for example,100µM.
• 3. Annealing components of 25 μM Final Duplex Concentration for reference:

Nuclease-Free water	12 µl
Annealing Buffer (5X)	8 µl
crRNA oligo (100µM)	10 µl
tracrRNA oligo (100µM)	10 µl
Total Volume for Annealing	40 ul

• 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 3-6pmol of Cas9 protein and 3-6 pmol of crRNA:tracrRNA are sufficient for each gene editing reaction.

• How do I use the HPRT positive control? Read More »

CRISPR RNA/Cas9 Protein 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