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GeneArt™ Platinum™ Cas9 Nuclease (1 µg/µL) - FAQs

View additional product information for GeneArt™ Platinum™ Cas9 Nuclease (1 µg/µL) - FAQs (B25642)

7 product FAQs found

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.

What is the suggested molar ratio of IVT gRNA to Cas9 protein?

The molar ratio of IVT gRNA to Cas9 protein should be approximately 1.2:1.

How should the Cas9/gRNA ribonucleoprotein (RNP) complex be transfected into cells?

We recommend using the Neon Transfection System or the Lipofectamine CRISPRMAX Cas9 Transfection Reagent.

What transfection methods do you recommend when working with your CRISPR products?

For transfecting of the Cas9 protein, we would recommend using the Neon transfection system or Lipofectamine CRISPRMAX Cas9 Transfection Reagent. For transfection of mRNA, we would recommend using Lipofectamine MessengerMAX Transfection Reagent. For transfection of CRISPR vectors, we would recommend using Lipofectamine 3000 Transfection reagent.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

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.