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查看更多产品信息 GeneArt® CRISPR Nuclease mRNA - FAQs (A25640)
85 个常见问题解答
Invitrogen GeneArt Precision TALs除了可用于基因删除、基因下调和整合之外，还可以用于基因激活。此外，该系统是基于一个蛋白-DNA系统，而CRISPR是基于一个RNA-DNA系统。TALs效应子可以用于靶向包括哺乳动物、细菌、酵母、植物、昆虫、干细胞以及斑马鱼在内的任何细胞的任何基因。最后，使用TAL系统时脱靶效应更低。请参考下列论文（http://www.sciencedirect.com/science/article/pii/S016816561500200X），文中作者比较了TALEs技术和CRISPR技术。
有一些方法可以帮助提高效率，例如，加入抗生素选择和/或使用流式细胞仪分选以富集被转染的细胞都会对提高效率有所帮助。
很不幸，PAM序列对于CRISPR基因编辑是必须的。但是，如果没有PAM序列可用时，您可以使用我们的Invitrogen GeneArt Precision TAL效应因子核酸酶系统。
可以，Neon体系可用于多重gRNA转染。
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。
我们已经在在多个细胞系中试用了我们的mRNA核酸酶CRISPR体系，其中包括：293细胞、HeLa细胞、U2OS细胞、HCT116细胞、小鼠neuro-2a细胞、小鼠ES细胞、iPS细胞、K562细胞、Jurkat细胞、CHO细胞和A549细胞。
创建多个针对您的目标区域的gRNAs，然后与GeneArt CRISPR核酸酶mRNA或GeneArt Platinum Cas9核酸酶共转染。要获得与Cas9 mRNA一起使用的gRNAs，使用带有U6启动子的GeneArt CRISPR Strings DNA或IVT的 gRNAs(使用GeneArt CRISPR Strings DNA，T7或GeneArt Precision gRNA 合成试剂盒生成)。对于Cas9蛋白，使用IVT的gRNAs(使用GeneArt CRISPR Strings DNA，T7或GeneArt Precision gRNA合成试剂盒生成)。
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。
可以，如果您使用目前的GeneArtCRISPR核酸酶载体，请留意对应的有限使用商标许可(LULL)。
不可以，对细菌来说，用于Cas9的PAM序列是特定的。GeneArt试剂盒中的Cas9来源于 Streptococcus pyogenes（酿脓链球菌）。
PAM具体是指前间区序列邻近基序，是Cas9成功结合到DNA上所必需的。GeneArt CRISPR试剂盒中Streptococcus pyogenes（酿脓链球菌）Cas9的PAM序列就是NGG。
将CRISPR-Cas9编辑复合物（DNA载体，mRNA或蛋白）以及修复模板共转染入细胞，其中修复模板中含有与目的序列高度同源的序列以及需要导入的DNA序列。这样，就可以通过HDR将特异性编辑（突变、插入等）引入基因组了。
可以通过GeneArt基因组切割检测实验检测切割效率。该实验通过可以识别错配的核酸内切酶来检测细胞内NHEJ修复过程中产生的插入和缺失(indel)。
Indel指的是基因组中的碱基插入或缺失，可以通过NHEJ或HDR修复过程引入细胞。
因为特定位点的切割效率取决于位点的易用性、染色质状态和序列，建议检测目标基因中的多个不同位点/区域。利用CRISPR-Cas9进行基因组编辑，对于不同的靶标，用户只需要改变19–20 bp的靶标特异性寡核苷酸。经过筛选细胞系并鉴别切割效率最高的序列/位点之后，可通过高特异性的Invitrogen GeneArt TALs（https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html）精确创建生物学相关的突变。
切割具有较高的精确度，在Cas9和gRNA复合物结合到靶向基因组序列后，在PAM (NGG)位点上游3个碱基的位置处发挥核酸酶活性。
对于mRNA，我们最早在转染24小时后开始观察到敲低效果，在48-72时后观察到更高的敲低效果。
Cas9仅瞬时表达，会随着时间和细胞分裂而消失。
也许可以，但我们的体系不是针对原核生物设计的，仅仅针对哺乳动物体系进行了优化。请咨询我们的CRISPR客户服务(custom.services@lifetech.com)详细咨询。
我们仅在哺乳动物体系（人和小鼠细胞）中检测过CRISPR体系。
是的，我们确实提供这项服务 (https://www.thermofisher.com/us/en/home/life-science/genome-editing/genome-engineering-services/cell-line-engineering-services.html)。
Invitrogen GeneArt CRISPR 核酸酶用户指南（https://tools.thermofisher.com/content/sfs/manuals/GeneArt_CRISPR_nuclease_mRNA_man.pdf）中的gRNA寡核苷酸设计策略阐释了设计gRNA需要定点插入新霉素抗性基因的方法。在新霉素抗性基因上连上位点特异的同源臂，就可以通过HDR插入新霉素抗性基因。
最好先针对前几个外显子进行设计（靠近启动子，导致转录提前终止）。由于gRNA效率取决于位点的易用性和该位点的染色质结构，通常建议设计和测试几个不同的靶向位点。从未经CRISPR处理的细胞中分离gDNA作为对照，通过 检测实验（https://www.thermofisher.com/order/catalog/product/A24372）可鉴别出非CRISPR相关的突变。标准免疫印迹分析是验证蛋白表达水平的最佳方法。
可以，将CRISPR技术与HDR结合使用将使之成为可能。
仔细设计crRNA用于靶向寡核酸以及避免与基因组上其他区域同源，是减少脱靶效应的关键。
HDR效率非常低，平均不到2%。
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）。
HDR（同源修复）和NHEJ（非同源末端连接）都是修复双链DNA损伤的细胞机制。不存在修复模板时，NHEJ用于双链断裂的连接，造成插入/缺失(indel)突变。HDR是另一种模板修复途径，可将序列复制到双链断裂处。因此，通过修复模板进行同源修复，可以将特定的核苷酸变化或者DNA片段引入到目标基因中。
我们建议对克隆进行分离，然后进行切割分析并对序列进行验证。
作为一种包括Cas9核酸内切酶和非编码的导向RNA（gRNA）的简单双组分体系，经过基因改造的II型CRISPR/Cas体系可用于在预先设定靶向的目标序列处切割基因组DNA。gRNA由两种分子组分：一种靶向互补的CRISPR RNA（crRNA）和一种辅助的反式激活crRNA（tracrRNA）。gRNA和PAM (NGG)基序引导Cas9核酸酶至基因组特定位置，形成复合物，之后局部链分离(R-loop)，Cas9核酸酶在PAM 位点上游3个碱基的位置形成一种双链DNA断裂（DSB）。因此，您既可以通过突变赋予靶标基因新的功能，实现敲除效果，也可以引入外来或合成的基因组序列，研究新的应用。
CRISPR也可灵活用于非编辑应用，例如基因调控或者RNAi相关的研究。Cas9核酸酶可以连接到不同的功能域（激活子或者抑制子）上，或者也可以设计gRNA用于直接切割miRNA。通过切割与修复机制的结合，TAL和CRISPR可直接编辑基因组产生永久的基因组变化（删除或移码突变），而且产生的基因敲除非常有效。而RNAi技术是通过作用于RNA（编码或非编码）从而下调或者完全关闭基因，是一种间接的方法。由于敲低水平取决于启动子的活性（与整合位点有关），即便在miRNA或shRNA体系稳定表达的情况下，RNAi技术也很难达到完全外显（即shRNA:mRNA的比例）的效果。
由于CRISPR-Cas系统高度灵活并且特异靶向，通过操作和调配，可成为基因组编辑的有力工具。CRISPR-Cas技术可用于多种真核生物的靶向基因切割和基因编辑，并且由于CRISPR-Cas系统中核酸内切酶切割的特异性由RNA序列导向，您也可以设计导向RNA序列并且将其与Cas核酸内切酶一起递送到靶标细胞中，从而在基因组任何位点进行编辑。
CRISPR指的是成簇的、规律间隔的短回文重复序列。在多种宿主生物体中，CRISPR-Cas（CRISPR-相关的）系统被用于基因组编辑。
CRISPR-STOP is a method of inserting STOP codon sequences to generate knockouts.
Please refer to the following article: CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.
Find additional tips, troubleshooting help, and resources within our Genome Editing Support Center.
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.
Our CRISPR products are optimized for mammalian systems, and have not been optimized for plant systems. However, we know researchers are doing that kind of work. While we have not tested our CRISPR products for plants, our new protein format would be ideal since it does not need to be translated and transcribed in the cell, so no plant-specific promoters required.
The Precision gRNA Synthesis Kit (Cat. No. A29377) includes primers for synthesis of gRNA targeting safe harbor locus HPRT. We also have a fully processed, fully validated HPRT gRNA with GCD primers for confirmation of cleavage available from our custom services team.
If you are delivering a single-stranded oligonucleotide on its own, we recommend using Lipofectamine RNAiMAX Transfection Reagent. However, Lipofectamine MessengerMAX Transfection Reagent can be used for genome editing purposes when Cas9 mRNA is co-delivered with a sgRNA and a single-stranded or double-stranded donor template.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Lipofectamine MessengerMAX Reagent may be used for both single gRNA delivery or multiplexing (gRNA for multiple targets) purposes with high transfection efficiency when used with GeneArt Cas9 mRNA. For a detailed protocol, please refer to the GeneArt Cas9 mRNA manual (https://www.thermofisher.com/order/catalog/product/A25640?ICID=search-a25640). We recommend using either the Invitrogen GeneArt Genomic Cleavage Detection Kit (Cat. No. A24732) or Invitrogen GeneArt Genomic Cleavage Selection Kit (Cat. No. A27663) for mutant analysis.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Lipofectamine MessengerMAX Reagent demonstrates low toxicity and high transfection efficiency for all cell types (easy or difficult-to-transfect, primary, and stem cells).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Lipofectamine MessengerMAX Regent is an excellent reagent for co-delivery of Invitrogen GeneArt CRISPR Cas9 mRNA with in vitro transcribed gRNA for geneome editing purposes. Lipofectamine MessengerMAX Reagent has the added benefit of flexibly delivering short dsDNA or HDR templates (0.5-1 kb) which can be ordered through the Invitrogen GeneArt Strings services.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Lipofectamine MessengerMAX Transfection Reagent is an animal origin-free transfection reagent, especially formulated for the delivery of mRNA, small RNA (eg.,CRISPR IVT gRNA, siRNA, or miRNA), and short dsDNA or HDR templates (0.5-1 kb). Lipofectamine MessengerMAX Reagent is an excellent reagent choice for CRISPR-mediated genome editing applications.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Invitrogen GeneArt Precision TALs, in addition to gene deletion, down-regulation and integration, can also be used for gene activation. Additionally, the system is based on a protein-DNA system, in contrast to CRISPR, which is based on a RNA-DNA system. TALs can be used to target any gene in any cell, including mammalian, bacterial, yeast, plants, insect, stem cells and zebrafish. Lastly, off-target effects are low when using the TAL system. Please refer to the following paper (http://www.sciencedirect.com/science/article/pii/S016816561500200X) where the authors compared TALs and CRISPR technology.
There are several ways to increase efficiency, for instance, adding antibiotic selection and/or FAC sorting to enrich for the transfected cells will both help.
PAM is a necessary requirement for CRISPR gene editing. However, in its absence, we recommend engineering a TAL effector to edit your desired gene efficiently. We offer GeneArt PerfectMatch TAL effectors. These are TAL effector nucleases that remove the 5´ base constraint and can be designed to target any desired sequence within the genome. Please go here for further details: https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html
Yes, the Neon system does work for multiple gRNAs transfected at the same time.
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 have tried our mRNA nuclease CRISPR system in multiple cell lines including: 293, HeLa, U2OS, HCT116, mouse neuro-2a, mouse ES cells, iPS cells, K562, Jurkat cells, CHO cells, and A549.
Create multiple gRNAs targeting the targets of your choice, followed by co-transfection with GeneArt CRISPR Nuclease mRNA or GeneArt Platinum Cas9 Nuclease. To make the gRNAs for Cas9 mRNA, use GeneArt CRISPR Strings DNA, U6 or IVT gRNAs (generated using either GeneArt CRISPR Strings DNA, T7 or the GeneArt Precision gRNA Synthesis Kit). For the Cas9 protein, use IVT gRNAs (generated using either GeneArt CRISPR Strings DNA, T7 or the GeneArt Precision gRNA Synthesis Kit).
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.
Yes, if you use the current Invitrogen GeneArt CRISPR nuclease vectors the respective Limited-Use Label Licenses (LULLs) will apply.
No, the PAM sequence is unique to the bacterial species that was used to create the Cas9. In the Invitrogen GeneArt kits, we derived Cas9 from Streptococcus pyogenes.
PAM stands for the protospacer adjacent motif. It is necessary for Cas9 to bind to the DNA successfully. The PAM sequence for the Streptococcus pyogenes Cas9 in the Invitrogen GeneArt CRISPR kits is NGG.
With the CRISPR-Cas9 editing complex (DNA vector, mRNA or Protein), co-transfect a DNA repair template that contains high homology to the sequence of interest along with the desired sequence you would like to introduce into the DNA. By doing so HDR can occur, and your specific edits (mutation, insertion, etc.) can be incorporated into the genome.
Cleavage efficiency can be detected using the Invitrogen GeneArt Genomic Cleavage Detection Assay. This assay relies on mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.
An indel refers to the genomic insertion or deletion of bases, which are incorporated during either cellular NHEJ or HD repair mechanisms.
Since cleavage efficiency at a particular locus depends on the accessibility of the locus, chromatin state, and sequence, it is advisable to test multiple different loci/regions within a gene of interest. With CRISPR-Cas9-mediated genome editing, for each target of interest the user needs only to change the 19-20 bp target-specific oligo. After the cell lines have been screened and the sequence/locus with the highest cleavage efficiency has been identified, the biologically relevant mutations can be precisely created with high-specificity Invitrogen GeneArt TALs (https://www.thermofisher.com/us/en/home/life-science/genome-editing/geneart-tals.html).
Cleavage is precise, and, after binding of the Cas9 and gRNA complex to the target genomic sequence, the nuclease activity occurs 3 bases upstream of the PAM (NGG) site.
This would depend upon the half-life of the particular transcript in your cell. We typically start seeing reduction in mRNA levels as early as 24 hrs post transfection, with further reduction after 48-72 hrs. Hence, we recommend performing the genomic cleavage detection assay 48-72 hours post transfection.
Cas9 is transiently expressed and will therefore disappear over time with successive cell divisions.
Yes, it is possible but our system is not for prokaryotes, and has only been optimized for mammalian systems. Please also consult our CRISPR custom services for further inquiries (custom.services@lifetech.com).
We have only tested these in mammalian systems (human and mouse cells).
Yes, we do offer this service (https://www.thermofisher.com/us/en/home/life-science/genome-editing/genome-engineering-services/cell-line-engineering-services.html).p>
The gRNA oligo design strategy in the Invitrogen GeneArt CRISPR Nuclease User Guide (https://tools.thermofisher.com/content/sfs/manuals/GeneArt_CRISPR_nuclease_mRNA_man.pdf)describes how you can design the guide RNA to target the locus in which the neomycin cassette should be inserted. The cassette (neomycin) can be inserted via HDR, in which case the neomycin cassette should contain locus specific homology arms.
The first few exons would be best (closer to the promoter, resulting in premature transcript termination). Since the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location, it is advisable to design and test a few target sites. Non-CRISPR-related mutations may be identified using gDNA isolated from non-CRISPR-treated cells as a control and performing a Invitrogen GeneArt Genomic Cleavage Detection Assay (https://www.thermofisher.com/order/catalog/product/A24372). Standard western blot analysis is a good measure for the verification of protein levels.
Yes, this should be possible using CRISPR technology combined with HDR.
Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.
HDR efficiency is very low, on average less than 2%.
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.
Both HDR (homology directed repair) and NHEJ (non-homologous end joining) are cellular mechanisms through which double-stranded DNA lesions are repaired. When a repair template is not present, NHEJ occurs to ligate double-stranded breaks, leaving behind insertion/deletion (indel) mutations. HDR is an alternative repair pathway in which a repair template is used to copy the sequence to the double-stranded break. You can, therefore, introduce specific nucleotide changes or DNA fragments into your target gene by using HDR with a repair template.
Clonal isolation and a combined cleavage analysis and sequence verification of the edited clone is advisable.
As a simple two-component system that includes the Cas9 endonuclease and a noncoding guide RNA (gRNA), the engineered Type II CRISPR/Cas system can be leveraged to cleave genomic DNA at a predefined target sequence of interest. The gRNA has two molecular components: a target-complementary CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). Both the gRNA and the PAM (NGG) motif guide the Cas9 nuclease to a specific genomic sequence to form a complex, followed by local strand separation (R-loop), at which the Cas9 nuclease creates a double-stranded DNA break (DSB) 3 nucleotides upstream from the PAM site. As a result, you may bring new functionality to the gene of interest via mutations, create knockouts, or introduce nonnative or synthetic genomic sequences to investigate novel applications.
CRISPR also allows for non-editing application flexibility such as gene regulation or RNAi-related studies. The Cas9 nuclease may be tethered to different functional domains (activators or repressors) or the gRNA may be designed to directly cleave miRNA.
TAL and CRISPR directly edit the genome by a combined cleavage and repair mechanism to impart permanent genomic change (deletion or frameshift mutation), and the resulting gene knockouts are very efficient. RNAi technology, on the other hand, is an indirect method in either down-regulating or shutting down a gene completely through direct interaction with RNA (coding or noncoding). Even in the case for stably expressed miRNA or shRNA systems, it may be difficult to effect complete penetrance (i.e., shRNA:mRNA ratio) since knock-down levels are dependent on the activity of the promoter (related to integration location).
With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell.
CRISPR stands for clustered regularly interspaced short palindromic repeat; CRISPR-Cas (CRISPR-associated) systems are used for genome editing in various host organisms.