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Invitrogen™ GeneArt™ CRISPR Nuclease mRNA

Catalog No. A29378
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A29378 15 μg
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For research use only. Not for use in diagnostic procedures.
Catalog No. A29378 Supplier Invitrogen™ Supplier No. A29378
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For research use only. Not for use in diagnostic procedures.

Description

Includes

1 vial (15 μg) GeneArt Platinum Cas9 Protein

Superior quality GeneArt CRISPR Nuclease mRNA is formulated to improve product performance.

GeneArt CRISPR Nuclease mRNA is formulated for superior quality and improved product performance. The mRNA format is ready to transfect and eliminates time-consuming cloning required when using CRISPR vector systems.

This wild-type Cas9 mRNA has two nuclear localization signals (NLSs) which has been shown to be more efficient at targeting the Cas9 protein to the nucleus (1).

The mRNA format is ready to transfect and eliminates time-consuming cloning required when using CRISPR vector systems. Cas9 mRNA is co-transfected with a target-specific guide RNA (gRNA) to direct the Cas9 protein to the intended genome locus to create a double-stranded break.

The complete RNA format is a smaller payload than plasmid-based Cas9 systems for improved delivery into the cell and better genome editing efficiency. Additionally, the Cas9 mRNA can be used in multiplex approaches with more than one gRNA. Use this approach to determine which gRNA sequence works best for a particular target or to edit multiple genomic loci with one transfection. Use our GeneArt Precision gRNA Synthesis Kit or GeneArt CRISPR Strings to make your target specific gRNA.

GeneArt CRISPR Nuclease Vector product features include:
  • Fewer steps with no annealing, cloning, or vector required for screening experiments
  • Smaller payload for improved delivery and better genome editing efficiency
  • T7 promoter option to improve cleavage in difficult to transfect cells
  • Easier for multiplexed gene editing
  • Easier delivery by microinjection
For research use only. Not for use in diagnostic procedures.

Certifications

CoA

Recommended Storage

  • Store in ultra-cold freezer (-68 to -85°C).
  • Product may be stored short-term in freezer (-5 to -30°C).

Specifications

Content And Storage 1 vial CRISPR Nuclease mRNA

Store in ultra-cold freezer (-68° to -85°C).
Product may be stored short-term in freezer (-5° to -30°C).
Format Tube
Final Product Type RNA
Product Line GeneArt
Product Type Crispr Nuclease MRNA
Quantity 15 μg
Shipping Condition Dry Ice
Volume (Metric) 15 μg

Frequently Asked Questions (FAQs)

How do I check for off-target effects in my CRISPR-modified cell lines?

The only complete way to confirm that there are no off-target effects is to sequence the entire genome of your cell. Alternatively, a less thorough means of checking for off-target editing is to perform targeted sequencing of sequences with the highest probability of off-target effects (i.e., most similar to your CRISPR target region).

How many guide RNAs do you recommend designing against my desired edit locus?

A single guide RNA (gRNA) is all that is required for targeting, but we do recommend testing 2-3 gRNAs against each locus being targeted for cleavage. Testing multiple gRNAs increases the chances of finding a gRNA with high editing efficiency, which will reduce the screening time required to identify the clone of interest.

How much Lipofectamine CRISPRMAX Reagent, Cas9 protein, and gRNA should I complex for gene editing?

Please see the Lipofectamine CRISPRMAX Reagent manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0014545_lipofectamine_crispermax_QR.pdf) for protocol details or contact techsupport@thermofisher.com.

Does your GeneArt CRISPR nuclease mRNA design include a fluorescent reporter or tag?

When the GeneArt CRISPR nuclease mRNA is used with the GeneArt Genomic Cleavage Selection Kit (Cat. No. A27663), both Cas9/gRNA ribonucleoprotein function and edited cells can be simultaneously confirmed by either OFP or CD4 selection.
Which transfection reagent should I use to deliver the GeneArt CRISPR nuclease mRNA?

Lipofectamine MessengerMAX Transfection Reagent is especially formulated for the delivery of both mRNA and gRNAs for all cell types (easy or difficult-to-transfect, primary, and stem cells).
How can I be sure that the GeneArt CRISPR nuclease mRNA was delivered to my cells?

We recommend using a positive gRNA control with the GeneArt CRISPR nuclease mRNA to ensure that your transfection reagent was efficiently delivered to your cells. We have a functionally validated in vitro transcribed HPRT gRNA control available from our Custom Services group (send an email to custom.services@lifetech.com). Either the GeneArt Genomic Cleavage Detection Kit (Cat. No. A24372) or GeneArtGenomic Cleavage Selection Kit (Cat. No. A27663) may be used to confirm genomic cleavage activity by Cas9 nuclease.
What is the GeneArt CRISPR nuclease mRNA?

GeneArt CRISPR nuclease mRNA is a ready-to-transfect, mammalian codon-optimized Streptococcus pyogenes Cas9 nuclease mRNA that is properly capped and polyA-tailed for stability and high expression. The smaller payload of GeneArt CRISPR nuclease mRNA allows for single or multiplex gRNA delivery for CRISPR-mediated genome editing applications. High genome editing efficiency can be achieved when both CRISPR mRNA and gRNA are delivered with Lipofectamine MessengerMAX Transfection Reagent.
What is the suggested ratio of Cas9 mRNA to IVT gRNA?

We recommend starting at a ratio of 0.5 µg of Cas9 mRNA and 50 ng of each IVT gRNA per well in a 24-well format. You should determine the optimal ratio for your particular cell line via a dose-response study.

What reagent should I use to transfect the Invitrogen GeneArt CRISPR Nuclease mRNA?

We recommend Invitrogen Lipofectamine MessengerMAX reagent.

I have my own system to express gRNA. Can I use it with the GeneArt CRISPR Nuclease mRNA system?

Yes, if you have your own system to make a gRNA with a S. pyogenes TRACR sequence, it is not necessary to order GeneArt CRISPR Strings DNA.

What are the advantages of the CRISPR mRNA system compared to your existing plasmid format?

The CRISPR mRNA system contains ready-to-transfect wild type Cas9 mRNA that circumvents the need for a cell type-specific promoter.
How efficient is the Cas9 mRNA format compared to the all-in-one CRISPR plasmid format?

In most cell types tested, this complete RNA format exhibits higher cleavage efficiency than the plasmid format. Additionally, the Cas9 mRNA format circumvents the need for cloning, has a smaller payload size, allows Cas9-to-gRNA dosage optimization, flexibility with multiplexing, and does not have any promoter constraints.

What is included in the Invitrogen GeneArt CRISPR Nuclease mRNA Kit?

The kit contains a ready-to-transfect wild type Cas9 mRNA for performing CRISPR-Cas9-mediated genome editing. The Cas9 mRNA can be used in experiments through two methods:

- Ready-to-transfect format: Cas9 mRNA is co-transfected directly with custom Invitrogen GeneArt CRISPR U6 Strings DNA or other synthetic gRNA expression cassettes.

- Complete RNA format: Cas9 mRNA is co-transfected with in vitro transcribed gRNA. In vitro transcribed gRNA can be generated from Invitrogen GeneArt CRISPR T7 Strings DNA or other custom templates.

Following transfection, the Cas9 protein (generated by the mRNA) is directed by the crRNA sequence of the gRNA to the encoded genomic locus to perform the desired genome editing.

When should I use the Invitrogen GeneArt CRISPR Nuclease Vector system as opposed to the Invitrogen GeneArt CRISPR Nuclease mRNA system?

We recommend using the Invitrogen GeneArt CRISPR Nuclease Vector system when working with mammalian cells and in scenarios where there are no promoter constraints. We recommend using the Invitrogen GeneArt CRISPR Nuclease mRNA system when working with difficult-to-transfect cell lines, microinjections and for multiplexing.

Can you provide suggestions on how to introduce a fragment into the genome or important sequence by HDR (homology directed repair)?

Create a double-stranded DNA break using the GeneArt CRISPR Nuclease Vector (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-nuclease-vector.html), while simultaneously transfecting your plasmid-based donor repair template. Your donor repair template plasmid will contain the sequence you wish to introduce that is flanked by at least 500 bp (or more) of sequence, which results in efficient homologous recombination of your sequence.

What is most efficient for HDR (homology directed repair), and what length is recommended for the homologous exogenous DNA, single-stranded oligos, double-stranded oligos, or large homologous arms (>1 kb) from a plasmid?

All of them may work, but for better efficiency, a longer homology arm is better (at least 500 bp (or more) on either side of the exogenous DNA). The homology length is dependent on the size of the fragment and will need to be tested. ssDNA may be error-prone or choose NHEJ. We offer the Invitrogen GeneArt Strings dsDNA fragments (1-3 kb) to assist with this type of application.

For Research Use Only. Not for use in diagnostic procedures.