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View additional product information for GeneArt™ CRISPR Nuclease mRNA - FAQs (A29378)
25 product FAQs found
对于24孔板规格，我们建议的起始比例为每孔0.5 µg Cas9 mRNA：50 ng IVT gRNA。建议您通过剂量反应实验确定对于特定细胞系的最佳比例。
我们建议采用Lipofectamine MessengerMAX试剂。
可以，如果您有自己的表达体系来产生带有S. pyogenes（酿脓链球菌） TRACR序列的gRNA，，就不必订购GeneArt CRISPR Strings DNA了。
CRISPR mRNA体系包含可直接转染的野生型Cas9 mRNA，没有细胞特异性启动子限制。
在大多数检测过的细胞类型中，相对于质粒形式，完整的RNA形式表现出更高的切割效率。另外，Cas9 mRNA形式避免了克隆的需要，负载更小，便于优化Cas9/gRNA剂量比，可以灵活地对多个靶点同时进行编辑，且不存在任何启动子相关的限制。
该试剂盒包含一个可直接转染的野生型Cas9 mRNA，可用于进行CRISPR-Cas9介导的基因编辑。Cas9 mRNA在各种实验中主要以两种形式应用：
可直接转染形式：Cas9 mRNA直接与定制的Invitrogen GeneArt CRISPR U6 Strings DNA或者其它方式合成的gRNA表达元件共转染。
完整的RNA形式：Cas9 mRNA与体外转录gRNA共转染。如果需要体外转录gRNA，模板可以采用Invitrogen GeneArt CRISPR T7 Strings DNA 或者其他定制模板。
在哺乳动物细胞以及没有启动子限制的情况下，建议使用GeneArt CRISPR核酸酶载体体系。而对于难转染的细胞系，显微注射以及需要同时靶向多个靶点时，建议使用GeneArt CRISPR核酸酶mRNA体系。
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。
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）。
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.