CRISPR sgRNAsNew!
GenScript CRISPR single-guide RNAs (sgRNAs) are 97-103 nt, fully customizable RNA oligonucleotides, allowing for greater flexibility in your CRISPR experiential design. Studies show that sgRNA has better stability than crRNA:tracrRNA when duplexed with Cas9, thus higher editing efficiency even in hard-to-transfect cells.
GenScript provides both unmodified sgRNA and modified sgRNAs. Modified sgRNAs with 2'-O-methyl and phosphorothioate modifications at the first three 5' and 3' terminal residues are recommended for better stability and editing efficiency.
*Deliver in as short as 8 business days.
| Product |
Length |
Quantity |
Pricing |
|
| GenCRISPR sgRNA |
97 to 103 nt |
4 nmol |
$259 |
|
| Modified GenCRISPR sgRNA (2'-O-methyl and phosphorothioate modifications at the first three 5' and 3' terminal RNA residues) |
97 to 103 nt |
4 nmol |
$359 |
|
CRISPR/Cas9 crRNAs
GenScript CRISPR RNAs (crRNAs) are short 36 nt RNA oligonucleotides which contain a short 20 nt sequence which guide the CRISPR/Cas9 complex to genomic targets for gene editing.
*Deliver in as short as 8 business days.
| Product |
Length |
Quantity |
Pricing |
|
| GenCRISPR crRNA |
20 nt |
2 nmol |
$95 |
|
| GenCRISPR crRNA |
20 nt |
10 nmol |
$125 |
|
| GenCRISPR crRNA |
20 nt |
20 nmol |
$225 |
|
CRISPR/Cas9 tracrRNAs
GenScript CRISPR/Cas9 trans-activating crRNAs (tracrRNAs) are 67 nt RNA oligonucleotides which together with the crRNA and Cas9 nuclease, form the activated CRISPR/Cas9 ribonucleoprotein complex.
*In-stock
| Product |
Quantity |
Pricing |
|
| GenCRISPR tracrRNAs |
5 nmol |
$95 |
|
| GenCRISPR tracrRNAs |
10 nmol |
$125 |
|
| GenCRISPR tracrRNAs |
20 nmol |
$225 |
|
Nuclease-Free Annealing Buffer
For annealing tracrRNA and crRNA.
| Product |
Concentration |
Quantity |
Pricing |
|
| Nuclease-Free Annealing Buffer |
5X |
100 µL |
$5 |
|
| Nuclease-Free Annealing Buffer |
5X |
1 mL |
$10 |
|
HPRT Primer Mix
Nuclease-Free Annealing Buffer
| Product |
Quantity |
Pricing |
|
| Nuclease-Free Annealing Buffer |
2 nmol |
$10 |
|
Cas9 Protein
Cas9 protein is the CRISPR-associated nuclease which enzymatically cleaves DNA and enables gene editing. GenScript Cas9 contains a N-terminal nuclear localization signal (NLS) and C-terminal NLS, for optimized nuclear compartmentalization.
*In-stock
| Product |
Quantity |
Pricing |
|
| GenCRISPR Sp Cas9 Nuclease 2NLS |
10 µg, 1µg/µL |
$95 |
|
| GenCRISPR Sp Cas9 Nuclease 2NLS |
25 µg, 1µg/µL |
$195 |
|
CRISPR/Cas9 Control Kit
GenScript offers pre-duplexed human HPRT crRNA:tracrRNA as positive controls for your CRISPR experiments. HPRT (hypoxanthine phosphoribosyltransferase) is a housekeeping gene and commonly used control has already been pre-validated for efficient CRISPR cleavage. The kit comes with an HPRT primer mix for PCR analysis.
*In-stock
| Product |
Pricing |
|
| GenCRISPR/Cas9 Control Kit |
$195 |
|
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:
- Incubate the crRNA and tracrRNA with the annealing buffer
- 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.
- 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 Ribonucleoprotein (RNP) User Manual
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.
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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.
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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 |
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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.
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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