GeneArt™ CRISPR Nuclease Vector with OFP Reporter Kit - FAQs

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103 product FAQs found

相比CRISPR系统，TAL基因组编辑技术的优点是什么？

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。

在我的筛选平板上仅出现很少的氨苄抗性菌落。可能的原因是什么？有什么建议吗？

请看下面的原因和解决方案：

- 单链（ss）寡核苷酸设计不正确：确保每个ss寡核苷酸均在3’末端包含用于克隆到GeneArt CRISPR核酸酶载体上的5个核苷酸，比如：正义链3’末端包含GTTTT，反义链3’末端包含CGGTG。
- 双链（ds）寡核苷酸被降解：将5 nM ds寡核苷酸存储于1X寡核苷酸退火缓冲液中，避免反复冻融；将5 nM ds寡核苷酸储存液分装并保存于–20°C。
- 寡核苷酸退火反应效率过低：确保根据实验说明进行退火反应。
- 如果环境温度>25°C，则需要在25°C温箱内进行退火反应。

我在对载体进行测序以确认插入片段已插入并且序列正确时遇到一些问题。有什么建议吗?

请看下列建议：

•使用高质量的、纯化的质粒DNA进行测序。我们建议使用Invitrogen PureLink HQ Mini质粒纯化试剂盒纯化DNA。向测序反应中加入DMSO至终浓度5%。
•提高用于测序反应的模板量（可提高至通常浓度的两倍）。
•在测序反应中使用7:1摩尔比的dITP:dGTP。

怎样才能提高效率？

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。

在我的目标区域附近没有PAM NGG序列。我该怎么办？

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。

可否利用Invitrogen Neon转染体系进行多重转染？

可以，Neon体系可用于多重gRNA转染。

Cas9 mRNA:IVTgRNA的最佳比例是多少？

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。

我对多重敲除非常感兴趣，请问如何采用GeneArt CRISPR进行多重敲除？

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)。

能否在已有的CRISPR-Cas9质粒中插入筛选标志物，如新霉素或者荧光标志物？

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)。

某些文献中使用了其他的PAM序列，如NNNGATT、NNAGAA或NAAAAC。能否使用这些序列替代NGG？

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）。

什么是PAM序列？

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。

我对使用CRISPR体系通过HDR而不是NHEJ修复来修饰特定的基因感兴趣，应该如何操作？

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。

如何判断我的CRISPR基因组编辑实验是有效的？

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)。

OFP激发和发射光的波长是多少？应选用哪种激光和发射滤光片进行检测？

OFP的激发峰为548 nm，发射峰为560 nm。我们建议采用488nm激光进行高效的激发，建议采用530/30、647/26和603/48发射滤光片进行检测。

GeneArt CRISPR载体中用的是什么启动子驱动gRNA的表达？

gRNA表达由Pol III U6启动子驱动。

GeneArt CRISPR核酸酶载体试剂盒的质保期为多久？

恰当储存的情况下，所有试剂可保证6个月内性质稳定。载体、缓冲液、对照寡核苷酸、质粒，水以及测序引物应储存在–20°C。

你们推荐采用哪种试剂在培养的细胞中转染CRISPR质粒？

转染方法因细胞类型而异。为了在大多数的哺乳动物细胞系中实现高效转染，我们推荐采用基于阳离子脂质体的 Invitrogen Lipofectamine 3000试剂。

在将DNA转染到真核生物细胞中之前，应如何纯化我的DNA？

建议使用 Invitrogen PureLink HiPure Plasmid Miniprep试剂盒来制备高质量的DNA。请务必确保DNA中不含苯酚或者氯化钠污染。

在连接和转化后，如何分析我的转化子？

通过测序确定插入到阳性转化子中的双链寡核苷酸的一致性。分析每一个CRISPR核酸酶重组子以确定：

•ds寡核苷酸插入物是存在的，并且方向正确；
•插入的双链寡核苷酸序列是正确的

注：由于双链寡核苷酸插入物太小，不建议进行限制性酶切分析。

连接之后，能否使用我自己的感受态细胞来转化GeneArt CRISPR核酸酶载体？

可以，我们同时提供含有或不含感受态细胞的试剂盒。当然，该载体优化时采用的是Invitrogen One Shot TOP10化学感受态，这种感受态专用于高效克隆、质粒扩增以及高拷贝质粒的稳定复制。请注意，使用不同基因型的感受态细胞可能会降低克隆效率，并且会产生较高比例的未插入载体。

如何设计和订购CRISPR寡核苷酸用于核酸酶载体构建？

可以先设计两条单链DNA寡核苷酸 (24–25 bp)，一条编码靶向特异性crRNA（正向链寡核苷酸），另一条是其互补的序列（反向互补链寡核苷酸）。然后您可以简单地通过退火制备双链寡核苷酸，将其克隆到试剂盒中提供的线性化的载体中。以下是我们提供的一些指导准则：

•长度：靶序列的长度在19-20nt之间，3´末端紧邻NGG(PAM)序列。通过U6PolIII启动子启动转录所需的5´G已经包含在载体的突出末端中，不需要包含在靶向序列中。注：不要在寡核苷酸引物中引入PAM序列。
•同源性：确保靶向序列与其他基因没有明显的同源性，否则可能会增加脱靶效应。最近发表的研究成果表明，gRNA-Cas9复合物可以容忍1–3个或者更多的错配，具体因其在gRNA中的位置而异。有关如何选择靶向序列的更多信息，请参考文献（Fu et al., 2013; Mali et al., 2013）。
•方向：您选择的靶向序列可能编码靶向位点的有义序列，也可能编码其反义序列。因此，您制备出的CRISPR RNA可能有两个朝向，在其3´端都有PAM序列。

带有CD4报告基因的GeneArt CRISPR核酸酶载体试剂盒中的CD4报告基因有什么好处？

转染了带有CD4报告基因的GeneArt CRISPR核酸酶载体的细胞群可以使用流式或者Invitrogen Dynabeads CD4磁珠进行富集。

带有OFP报告基因的GeneArt CRISPR核酸酶载体试剂盒中的OFP报告基因有什么好处？

OFP报告基因表达的荧光方便追踪转染效率，同时支持通过FACS法分选/富集表达Cas9和CRISPR的细胞。

哪种启动子可以驱动Cas9的表达？

我们的载体包含了一个CMV启动子，可驱动Cas9核酸内切酶的表达。

收到CRISPR寡核苷酸后，我该如何对它进行重悬？

可以用无核酸酶的水或TE缓冲液进行重悬，但如果您要进行退火反应，请使用合适的寡核苷酸退火缓冲液进行退火。随后，请用终浓度1X的寡核苷酸退火缓冲液对引物做系列稀释。

在设计用于CRISPR实验的寡核苷酸时，你们推荐采用何种等级或纯度？寡核苷酸需要磷酸化处理吗？

5’端非磷酸化的脱盐纯化的寡核苷酸是可以的。当然，HPLC或更高纯度的寡核苷酸将会增加双链寡核苷酸克隆到GeneArt CRISPR核酸酶载体的效率。

使用GeneArt CRISPR核酸酶载体有哪些优点？

该体系使得您可以通过单个质粒，以序列特异性方式编辑和改造所选的基因组位点。我们提供两种载体，分别表达OFP报告基因和CD4，便于通过流式或者InvitrogenDynabeads技术筛选和富集细胞。这两种试剂盒都包括一体化的载体，可以同时表达非编码导向RNA（包括crRNA和tracrRNA）和Cas9核酸内切酶。

Invitrogen GeneArt CRISPR核酸酶载体和Invitrogen GeneArt CRISPR核酸酶mRNA体系分别在什么情况下使用？

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。

什么是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。

在使用TALs之前，你们建议采用CRISPR做为筛选工具，这是出于什么考虑呢？

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。

切割活动的精确度如何？

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。

你们转染后多久对mRNA和蛋白表达敲低进行检测？

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发挥其核酸内切酶活性之后会发生什么？

Cas9仅瞬时表达，会随着时间和细胞分裂而消失。

CRISPR-Cas技术能用于原核生物基因改造吗？

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。

除哺乳动物类体系，你们有没有在其它宿主中检测过CRISPR体系？

我们仅在哺乳动物体系（人和小鼠细胞）中检测过CRISPR体系。

你们提供利用CRISPR制备定制细胞系的服务吗？

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)。

我希望设计一个gRNA用于插入筛选标记（例如新霉素），应该如何操作？

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。

我正在尝试靶向一种分子量较大的多功能蛋白，该如何设计sgRNA位点？怎样才能核实编辑是否发生？

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。

CRISPR技术可用来引入大分子序列吗，比如GFP或IRES?

可以，将CRISPR技术与HDR结合使用将使之成为可能。

我很担心脱靶效应，有没有好办法来应对？

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。

在双链断裂后HDR的效率是多少？

HDR效率非常低，平均不到2%。

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）。

NHEJ和HDR介导的修复有哪些区别？

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。

在不同细胞中特定位点发生的插入缺失是否完全相同？是否应该在扩大和富集群体之前引入克隆步骤？

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。

CRISPR-Cas体系包含哪些组分？

有3种组分：

•Cas核酸酶Cas9（一种双链DNA核酸内切酶）
•一种靶向互补crRNA
•一种辅助的tracrRNA

Invitrogen GeneArt CRISPR核酸酶载体的crRNA和tracrRNA同时表达形成向导RNA，模拟细菌体系中自然存在的crRNA-tracrRNA嵌合体。

Invitrogen GeneArt CRISPR-Cas体系的工作原理是什么？

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。

与载体形式的稳定表达shRNA相比，通过TAL或CRISPR产生基因敲除的优势是什么?

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。

CRISPR技术有何用途？

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。

何为CRISPR和 CRISPR-Cas？

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。

What is CRISPR-STOP?

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.

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.

Do you have CRISPR products optimized for plants?

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.

What are the benefits of using TAL/TALEN-based genome editing compared to your CRISPR system?

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.

I am using the Invitrogen GeneArt CRISPR Nuclease Vector Kit and am getting very few ampicillin-resistant colonies on my selection plate. What could be the cause of this? Do you have any suggestions?

Here are possible reasons and solutions:

- Single-stranded (ss) oligonucleotides designed incorrectly; Make sure that each ss oligonucleotide contains the 5 nucleotides on the 3′ end required for cloning into the Invitrogen GeneArt CRISPR Nuclease Vector: ie., Top strand includes GTTTT on the 3′ end; Bottom strand includes CGGTG on the 3′ end.

- Double-stranded (ds) oligonucleotides were degraded; Store the 5 nM ds oligonucleotide stock in 1X Oligonucleotide Annealing Buffer. Avoid repeated freeze/thaw cycles; aliquot the 5 nM ds oligonucleotide stock and store at -20°C.

- Oligonucleotide annealing reaction inefficient; Ensure that the annealing reaction was performed as directed. If ambient temperature is >25°C, incubate the annealing reaction in a 25°C incubator.

I am using the Invitrogen GeneArt CRISPR Nuclease Vector Kit. I am having problems sequencing my construct to verify that my ds oligonucleotide insert is present and has the correct sequence. What do you recommend?

Please see the following recommendations:

- Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen PureLink HQ Mini Plasmid Purification Kit. Add DMSO to the sequencing reaction to a final concentration of 5%.
- Increase the amount of template used in the sequencing reaction (up to twice the normal concentration).
- Use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.

How can I increase my efficiency of genome editing using the CRISPR-Cas9 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.

I am interested in using the CRISPR-Cas9 technology for genome editing. However, there is no 5’ NGG (PAM) sequence close to my locus of interest. What can I do?

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

Can I perform multiplexed transfection of CRISPR gRNAs using the Invitrogen Neon Transfection System?

Yes, the Neon system does work for multiple gRNAs transfected at the same time.

What is the suggested ratio of Cas9 mRNA to IVT gRNA?

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.

I am interested in multiplexing. How can I do this using the GeneArt CRISPR system?

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).

Is it possible to insert a selection marker, e.g., neomycin or a fluorescent marker, into a preexisting CRISPR-Cas9 plasmid?

Yes, if you use the current Invitrogen GeneArt CRISPR nuclease vectors the respective Limited-Use Label Licenses (LULLs) will apply.

I see other publications using different PAM sequences such as NNNGATT, NNAGAA, or NAAAAC. Can I use that instead of NGG?

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.

I am using the CRISPR-Cas9 technology. What is the PAM sequence?

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.

I am interested in using the CRISPR system to modify my gene of interest using HDR instead of NHEJ repair. How can I do this?

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.

How can I tell if my CRISPR genome editing experiment is working?

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.

What is the excitation and emission of OFP? What laser and emission filters should I use for detection?

OFP has a peak excitation of 548 nm, and emission of 560 nm. We recommend a 488 nm laser for efficient excitation. Standard 530/30, 647/26, and 603/48 emission filters are recommended for detection.

What promoter is used to drive gRNA expression in the Invitrogen GeneArt CRISPR vectors?

gRNA expression is driven by the Pol III U6 promoter.

For how long are the reagents in the Invitrogen GeneArt CRISPR Nuclease Vector kit guaranteed?

All reagents are guaranteed to be stable for 6 months when properly stored. The vector, buffers, control oligo, plasmid, water, and sequencing primers should be stored at -20°C.

What reagent do you recommend for transfection of the CRISPR plasmids for cell culture?

The method of transfection varies based on cell type. For high-efficiency transfection in a broad range of mammalian cell lines, we recommend using the cationic lipid-based Invitrogen Lipofectamine 3000 Reagent.

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

What should I use to purify my CRISPR plasmid before transfection into eukaryotic cells?

We recommend using high-quality DNA prepared with the Invitrogen PureLink HiPure Plasmid Miniprep Kit. Ensure that DNA is free of contamination with phenol and sodium chloride.

After ligation and transformation of my CRISPR plasmid, how should I analyze my transformants?

Confirm the identity of the ds oligonucleotide insert in positive transformants by sequencing. Analyze each CRISPR nuclease construct to verify:

- That the ds oligonucleotide insert is present, and in the correct orientation
- That the ds oligonucleotide insert has the correct sequence

Note: Restriction analysis is not recommended due to the small size of the ds oligonucleotide insert.

Can I use my own competent cells to transform my Invitrogen GeneArt CRISPR nuclease vector after ligation?

Yes, we provide kits both with or without competent cells. However, the vectors were optimized using Invitrogen One Shot TOP10 chemically competent E. coli, designed for high-efficiency cloning and plasmid propagation and stable replication of high-copy number plasmids. Please note that using competent cells of different genotype may lower cloning efficiency and can also result in a higher proportion of vectors without insert

How do I design and order the CRISPR oligos for cloning into the nuclease vectors?

Start off by designing two single-stranded DNA oligonucleotides (24-25 bp), one encoding the target-specific crRNA (forward-strand oligonucleotide) and the other its complement (reverse complement-strand oligonucleotide). You can generate a double-stranded oligonucleotide suitable for cloning into the linearized vector provided in the kit by simply annealing the complementary oligonucleotides. Here are some guidelines:

- Length: Choose a target sequence ranging from 19 to 20 nucleotides in length that is adjacent to an NGG proto-spacer adjacent motif (PAM) sequence on the 3′ end of the target sequence. The 5′ G required for transcription initiation from the U6 Pol III promoter is already included in the vector overhangs and does not need to be included in the target sequence. Note: Do not include the PAM sequence in the oligonucelotide primers.
- Homology: Make sure that the target sequence does not contain significant homology to other genes, as this can increase off-target effects. Recently published work has shown that gRNA-Cas9 complexes can potentially tolerate 1-3 or more mismatches, depending on their location in the gRNA. Refer to published articles for more insights into choosing a target sequence (Fu et al., 2013; Mali et al., 2013).
- Orientation: You may choose a target sequence encoding either the sense sequence of the target locus or the antisense sequence. Thus, you can generate CRISPR RNA in two possible orientations, provided that it meets the PAM requirements on the 3′ end.

What are the benefits of using a CD4 reporter in the Invitrogen GeneArt CRISPR Nuclease Vector with CD4 Reporter Kit?

Populations of cells transfected with Invitrogen GeneArt CRISPR nuclease vectors containing CD4 reporters may be enriched using FACs or using Invitrogen Dynabeads CD4 magnetic beads.

What are the benefits of using an OFP reporter in the Invitrogen GeneArt CRISPR Nuclease Vector with OFP Reporter Kit?

The OFP reporter allows for fluorescence-based tracking of transfection efficiency, as well as FACS-based sorting/enrichment of Cas9- and CRISPR-expressing cells.

What promoter is driving expression of Cas9?

Our vectors contain a CMV promoter to drive expression of the Cas9 endonuclease.

When I receive my CRISPR oligonucleotide, what should I resuspend it in?

Either nuclease-free water or TE buffer is fine, but please anneal using the appropriate Oligonucleotide Annealing Buffer for your annealing reaction. Subsequently, please serially dilute your primers in a final concentration of 1X Oligonucleotide Annealing Buffer.

When designing my primers for the CRISPR oligonucleotide, what level or purity is recommended? And do the oligonucleotides need to be phosphorylated?

5′-nonphosphorylated desalted oligonucleotides are fine. However, HPLC or higher purity will increase the cloning efficiency of your double-stranded oligonucleotide into the Invitrogen GeneArt CRISPR Nuclease vector.

What are the advantages of using the Invitrogen GeneArt CRISPR Nuclease Vectors?

This system allows you to edit and engineer the genomic locus of your choice in a sequence-specific manner from a single plasmid. We offer two plasmids that can be used with these kits, with either the OFP reporter or CD4 ereporter, for ease of selection and enrichment by either FACs or Invitrogen Dynabeads technology. Both kits include all-in-one vectors that co-express both the noncoding guide RNA (including crRNA and tracrRNA) and the Cas9 endonuclease.

When should I use the Invitrogen GeneArt CRISPR Nuclease Vector system as opposed to the Invitrogen GeneArt CRISPR Nuclease mRNA system?

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.

What is an indel?

An indel refers to the genomic insertion or deletion of bases, which are incorporated during either cellular NHEJ or HD repair mechanisms.

Can you elaborate on your suggestion to use CRISPR as a screening tool before going to TALs?

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).

I am using the CRISPR-Cas9 technology. How precise is the cleavage event?

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.

I am using the CRISPR technology to knock out a gene. How long after transfection should I assess mRNA levels and protein knockdown?

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.

What happens to Cas9 after it performs its endonuclease activity?

Cas9 is transiently expressed and will therefore disappear over time with successive cell divisions.

Is it possible to use CRISPR-Cas technology for prokaryotic gene engineering?

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).

Have you tested the CRISPR system in hosts other than mammalian systems?

We have only tested these in mammalian systems (human and mouse cells).

Do you offer a service to generate custom cell lines using CRISPR?

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>

I am using the CRISPR technology. I would like to design a guide RNA (gRNA) to insert a selection cassette such as neomycin. How can I do this?

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.

I am trying to target a big and multifunctional protein using CRISPR technology. To what site should I design my sgRNA? How can I check that the editing occurred?

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.

Can I use CRISPR technology to introduce large sequences, such as GFP or IRES?

Yes, this should be possible using CRISPR technology combined with HDR.

I am using the CRISPR technology and am worried about off-target effects. What is the best way to combat this issue?

Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.

What is the efficiency of HDR (homology directed repair) after the double-stranded break?

HDR efficiency is very low, on average less than 2%.

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.

What is the difference between NHEJ- and HDR-mediated repair?

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.

Are the indel events that happen in a particular locus identical between the cells? Can we include a clonal step before expanding and enriching the population?

Clonal isolation and a combined cleavage analysis and sequence verification of the edited clone is advisable.

What comprises the CRISPR-Cas system?

There are 3 components:

- The Cas nuclease Cas9 (a double-stranded DNA endonuclease)
- A target-complementary crRNA
- An auxiliary tracrRNA

The crRNA and the tracrRNA of the Invitrogen GeneArt CRISPR Nuclease Vector are expressed together as a guide RNA that mimics the natural crRNA-tracrRNA chimera in bacterial systems.

How does the Invitrogen GeneArt CRISPR-Cas system work?

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.

What is the advantage of generating gene knockouts by TAL or CRISPR compared to vector-based stably expressed shRNA?

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).

What is CRISPR technology used for?

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.

What does CRISPR and CRISPR-Cas stand for?

CRISPR stands for clustered regularly interspaced short palindromic repeat; CRISPR-Cas (CRISPR-associated) systems are used for genome editing in various host organisms.