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View additional product information for GeneArt™ CRISPR Nuclease mRNA - FAQs (A29378)
16 product FAQs found
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).
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.
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.
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.
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).
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.
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.
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.
We recommend Invitrogen Lipofectamine MessengerMAX reagent.
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.
The CRISPR mRNA system contains ready-to-transfect wild type Cas9 mRNA that circumvents the need for a cell type-specific promoter.
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.
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.
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.
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.
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.