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

查看更多产品信息 GeneArt™ Platinum™ Cas9 Nuclease (1 µg/µL) - FAQs (B25642)

9 个常见问题解答

就如何利用HDR向基因组或者重要的序列中引入片段，你们有何建议？

利用 GeneArt CRISPR核酸酶载体（https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-nuclease-vector.html）引起双链DNA断裂，同时转染基于质粒的供体修复模板。您的供体修复模板质粒将会包含希望引入的序列并在两端具有至少500bp（或更长）的序列，从而实现序列的高效同源重组。

以下哪些最适合用于同源修复（HDR）：单链寡核苷酸、双链寡核苷酸或者来源于质粒的长同源臂（>1 kb）。对于同源外源DNA，你们建议的长度是多少？

都可以。但为了提高效率，最好使用较长的同源臂（在外源DNA的两端至少500 bp（或更长））。同源长度取决于片段长度且需要测试。ssDNA可能更容易出错或选择NHEJ途径进行修复。针对这种应用，我们提供Invitrogen GeneArtStrings dsDNA片段（1–3 kb）。

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.