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View additional product information for GeneArt™ Genomic Cleavage Detection Kit - FAQs (A24372)
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TALs或者TALENs是类似于转录激活因子的效应核酸酶蛋白，它们是天然存在的转录激活因子，由Xanthomonas spp.分泌到其植物宿主体内。 Invitrogen GeneArt TALs来源于细菌Xathomonas产生的TAL效应子，其DNA结合结构域包含不同数目的氨基酸重复序列。每个重复包含33-35个氨基酸并可识别一个单DNA碱基对。对DNA的识别是通过两个高变异氨基酸残基实现的，它们分别位于每个重复序列的第12和13位，被称为重复可变双残基（repeat-variable di-residues, RVD）。TAL效应子重复序列可以通过模块的形式组装，通过改变RVD而生成一个识别特定目标DNA序列的TAL蛋白。
我们在TAL MCS入门载体中提供一个多克隆位点替代效应子结构域。这一设计使得您可以向其中插入任何蛋白编码序列，并且可以根据序列特异性将您得到的TAL蛋白靶向到基因组的任何位置。我们还提供基因合成服务以生成任何您没有模板的效应子结构域。
虽然我们的Invitrogen GeneArt Precision TALs要求在5’末端有一个T并且在正向和反向TAL效应子之间有13–18 bp的间隔序列以便Fok1核酸酶进行正常配对，但是Invitrogen GeneArt PerfectMatch TALs允许靶向基因组任何序列并且消除了5’末端要求为T的限制。另外，两个效应子之间的最佳间隔序列为 15–16 bp。
19 bp结合结构域对于核酸酶而言效果更好。结合位点不需要是相同长度；但是对于核酸酶而言19 bp结合结构域效果最好。
我们观察到只要有3 bp的错配便会影响TAL的结合。我们建议设计序列和结合位点之间精确匹配。如果您需要获得设计上的帮助，请发邮件到geneartsupport@lifetech.com.
请看下表所列的常见问题、可能原因以及建议：
-条带弥散：裂解物浓度过高；将裂解物稀释2至4倍然后重复PCR反应。
-不同扩增子之间条带亮度有差异：不同样本内裂解物浓度不同；使用 Invitrogen PureLink PCR纯化试剂盒纯化PCR产物。欲比较样本间的效果并想获得最佳结果，请纯化PCR产物并在每个切割实验中使用相同量的DNA。每个反应使用50 ng至100 ng DNA已经足够。
-无PCR产物：PCR引物设计不佳或模板包含GC富集区域；重新设计引物，使其长度为18–22 bp，GC含量45–60%，并且Tm范围在52–58°C。对于GC富集区域，向50 µL反应体系内加入1–10 µL的360 GC Enhancer 并重复PCR扩增。
样本通常使用琼脂糖凝胶例如Invitrogen E-Gel EX 凝胶进行电泳，然后使用图像软件或微流控电泳进行分析。
是的，我们的GeneArt Genomic Cleavage Detection 试剂盒可以用于确定核酸酶对于给定位点的切割效率。
我们建议您将载体重悬于50 µL蒸馏水或10 mMTris-HCl (pH 8.0)缓冲液中，室温孵育1小时。轻轻吹打5-10次以重悬载体DNA。将重悬后的DNA存储在–20°C。
请发电子邮件到我们的团队，地址是outlicensing@lifetech.com.
不，我们推荐脂质体介导的转染或电转染。请查看相关文献或咨询您所用细胞系的提供者以获知优化的转染方法。
使用一对与Fok1内切核酸酶融合的GeneArt Precision TAL蛋白可以在基因组特定位点使双链断裂。使用一对Precision TAL蛋白可以降低脱靶效应。Fok1核酸酶结构域产生的双链切口将由两种细胞内源机制中的一种修复：非同源末端连接（nonhomologous end joining, NHEJ），或同源重组修复（homology-directed repair, HDR）。NHEJ倾向于错误修复并且在修复蛋白编码基因的编码区域时经常会引入移码突变，从而高效的导致基因沉默。同源DNA“供体序列”可以用于HDR修复以引入一段新的DNA序列。因此，与Fok1内切核酸酶融合的GeneArt Precision TAL蛋白可以用于诱导基因沉默或在基因组的特定位点精确插入一段设计好的DNA片段。
TAL形式以及考虑的情况如下：
- 截短体TAL Fok1：推荐用于哺乳动物细胞
- 天然TAL Fok1：推荐用于植物
- 天然激活子：在非哺乳动物系统内表现更好
- 截短体MCS：去除了内源激活子活性
所有TALs都作为入门克隆进行表达（兼容于Gateway克隆，两侧带有attL1和attL2位点）。
1. Fok1 TALs：
带有Fok1核酸酶的TALs可以被用于靶向沉默特定基因。
Fok1是一种来源于Flavobacteriumokeanokoites的二型限制性内切酶，它包含一个N端DNA结合结构域和一个C端非特异性DNA切割结构域。
Fok1作为成对的核酸酶发生作用，它在结合结构域指定的目标位点结合DNA双链并切割DNA。
2. VP16或VP64 TALs：
VP16或VP64均可以用于提高内源或重组基因的表达水平。
VP16是一种来源于单纯疱疹病毒的反式作用蛋白，它可以和宿主转录因子形成复合体以诱导早期基因的快速转录。
请根据标准的质粒DNA转染步骤进行操作。我们建议您使用我们的Lipofectamine 3000和Lipofectamine 2000试剂。
1.为目标DNA设计GeneArt TALs，然后将其克隆到Gateway入门载体或目的载体
2.体外验证TALs（可选）
3.用TAL表达载体转染细胞
4.TAL介导的目标DNA切割
5.使用GeneArt Genomic Cleavage Detection Kit分析切割效果
是的，KO和KI涉及到在需要的位点通过突变或删除遗传信息或插入新的信息编辑原始的遗传编码。尽管这一技术确实是对原始遗传信息的操作，但如果运用恰当,，它将非常有用，例如遗传操作酵母菌以生产胰岛素或者对细胞进行遗传工程操作以获得更经济和临床上更有价值的产品。
对于标准测试细胞系（如293，Hela等）导入mRNA和DNA的最佳方法是基于脂质体的转染。导入mRNA也可以降低转基因整合的风险。我们提供用于导入mRNA的产品包括Invitrogen Lipofectamine MessengerMAX 试剂，用于导入DNA的产品包括Invitrogen Lipofectamine 3000 试剂。对于干细胞，电转是最好的选择。
我们通常在收到您订单后2周内开始制造您的TAL载体。
通过仔细的设计可以构建非常特异的TALs。最近发表的文章显示TALs可以在很大程度上接受目标DNA序列内的1-3碱基的错配。
是的。如果基于病毒的导入方法是你的首选，那么我们建议使用腺病毒系统而非慢病毒系统进行TAL的导入。
TAL载体为3.3 kb。
最近由Prashant Mali发表在Nature Biotechnology上的研究（http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2675.html）指出TALENs可以接受1-2碱基的错配，但不太能接受大多数3碱基的错配。
我们提供N-TAL Fok1Gateway入门载体或Fok1 CMV启动子驱动表达的载体。
GeneArt Perfect Match TALs来源于GeneArt Precision TALs。GeneArt Perfect Match TALs采用了一个新的设计以去除5’端碱基限制，因此可以被设计为靶向基因组内的任何序列。它们在TAL效应子的N末端带有3个氨基酸的突变，从而将5’结合基序转变为一个通用结合基序，可以结合任意碱基：A，G，C或T。这些PerfectMatch TALs可以是带有Fok1核酸酶的Invitrogen Gateway入门载体，也可以是可在哺乳动物系统表达的带有CMV启动子的表达载体。PerfectMatch TALs的表现和最初的Precision TALs一样或更好。目前，GeneArtPerfectMatch TALs仅提供带有核酸酶功能的形式（Fok1核酸酶对），而Precision TALs提供核酸酶功能（Fok1核酸酶对），激活子功能（VP16或VP64），抑制子功能（KRAB），或者定制功能（MCS载体）。
我们使用 Invitrogen GeneArt Genomic Cleavage Detection试剂盒比较了针对HPRT位点设计的GeneArt Perfect Match和Precision TALs的切割效率，并且发现对于相同目标区域GeneArt Perfect Match TALs表现出和GeneArt Precision TALs相同或更好的性能。
GeneArtPerfectMatch TALs提高了设计TAL效应子的灵活性，并使得一对TAL效应子间保持15–16 bp的间隔成为可能，从而获得了最大的TAL效应子效率。
以下是我们的建议：
GeneArt Precision TALs允许构建针对18或24个碱基对的DNA靶标的TAL效应子功能性蛋白。
每个目标位点必须在5’末端之前有一个碱基T，因为TAL效应子蛋白包含一个保守的T-结合基序。该5’T不包含在被选作特异性靶标的18或24个碱基中。
一对核酸酶需要被设计为在互补DNA链上的各自目标位点之间有13-18 bp的间隔序列。
对于Invitrogen GeneArt PerfectMatch TALs而言，5’末端碱基没有限制。我们通过突变N端结构域以降低其对5’T的特异性从而研发出了第二代的TALs。因此，5’端出现任何碱基（T, G, C, 或A）都可以，且其性能表现和最初的GeneArt Precision TALs相当。我们建议您在设计一对核酸酶时，它们各自识别的目标位点之间应有一段15–16 bp的间隔序列。
因为存在强DNA结合基序和弱DNA结合基序，DNA结合结构域内每个结合基序对于TAL效应子结合DNA效率的贡献是不同的。A和T结合基序被认为属于“弱结合”基序，而C和G结合基序被认为是“强结合”基序。应该避免在结合结构域（不包括5’T）的5’末端出现5个连续的弱结合基序，也应避免5个连续的弱结合基序两侧不是C的情况。
我们建议您选择一个针对混合结合基序组成的结合结构域的TAL效应子以获得最佳结果。在活细胞中，DNA的开放程度也会影响TAL效应子的效率。染色质、DNA甲基化，和/或与DNA结合的蛋白都可能影响TAL的结合。
尽管启动子的结构各异，并且目前还缺乏特定的设计规则，但是我们建议将用于激活天然启动子的TAL转录因子设计在TATA-box的上游，或者在某些情况下将其置于转录起始位点下游。不建议将目标位点选择在跨TATA-box或其它已知转录因子结合位点区域。确保具有天然存在的ATG，并且没有转录出上游的其它ATG，否则可能干扰天然转录的起始。
请查看下列可用的效应结构域（https://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-precision-tals.html#table1）。
我们在您的订单确认后大约两周内可将一个经过验证和序列优化的克隆发出。
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。
1) 每个重复序列内的两个关键氨基酸和目标序列内的每个DNA碱基是一一对应的关系
2) 通过简单编码即可生成基因工程TAL蛋白
3) 比锌指蛋白更具可预测性
4) 结构域的模块化组装可以生成序列特异性的DNA结合蛋白
5) 可以通过编码对特定位点行使某种功能如：核酸酶，激活因子，抑制因子，染色体修饰因子
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。
待研究的基因组DNA位点在检测前必须进行PCR扩增。请遵照以下推荐的指导原则，确保最佳的扩增和后续检测效果：
•为了获得最佳的结果，请使用Tm>55°C的引物。
•引物设计长度要介于18–22 bp之间，且GC含量介于45–60%之间。
•为有效扩增，设计引物产生的扩增子长度要介于400到500 bp之间。
•引物设计时潜在的切割位点不能在扩增子的中心位置，同时检测反应需要能够产生两个明显的产物条带。
该检测体系所采用的基因组DNA是从转染了表达核酸酶的元件的细胞中提取的。切割之后，在细胞的修复机制下产生了插入和缺失，通过PCR对出现基因特异性双链断裂的位点进行扩增。PCR产物经过变性和再退火产生错配，因为含有插入/缺失的序列再退火后，不含插入/缺失的链会和含有插入/缺失的序列退火产生错配。错配随后被检测到并被检测酶切割，切割产生的条带即可通过凝胶电泳和条带的灰度值进行分析。
GeneArt基因组切割检测试剂盒为位点特异性双链DNA断裂提供了一种简单、可靠且快速的检测方法。该技术利用能识别错配的内切酶来检测细胞的NHEJ修复过程中引入的基因组插入或缺失(indel)。
GeneArt基因组切割筛选试剂盒
- 快速，活细胞检测
- 目测指示（荧光）
- 证明编辑工具有效
- 允许克隆富集
GeneArt基因组切割检测试剂盒
- 需要裂解细胞
- 可量化结果
- 对于阴性结果无法判断编辑工具是否有效
- 无富集能力
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。
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)。
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。
TALs or TALENs are transcription activator-like effector nuclease proteins that are naturally occurring transcriptional activators secreted by Xanthomonas spp. into their plant hosts. GeneArt TALs are derived from Xathomonas TAL effectors, the DNA-binding domain of which consists of a variable number of amino acid repeats. Each repeat contains 33–35 amino acids and recognizes a single DNA base pair. The DNA recognition occurs via 2 hypervariable amino acid residues at positions 12 and 13 within each repeat, called repeat-variable di-residues (RVDs). TAL effector repeats can be assembled in modular fashion, varying the RVDs to create a TAL protein that recognizes a specific target DNA sequence.
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.
We do offer a multiple cloning site sequence in the place of the effector domain sequence for our TAL MCS entry vector. This option allows you to insert any protein-coding sequence, and allows your resulting TAL protein to deliver the effector in a sequence-specific manner anywhere in the genome. We also provide gene synthesis services to generate any effector domain for which you don't have a template.
While our Invitrogen GeneArt Precision TALs required a T at the 5´end and 13-18 bp spacing between the forward and reverse TAL effectors for proper pairing of Fok1 nucleases, the Invitrogen GeneArt PerfectMatch TALs allow for targeting of any sequence across the genome and eliminates the 5´ T constraints. Additionally, the spacing between the two effectors is optimal at 15-16 bp.
The 19 bp binding domains perform better for the nucleases. The binding sites do not need to be the same size; however, best performance for the nucleases is with the 19 bp binding domains.
We have observed that as little as 3 bp mismatch affects the binding of the TALs. We recommend that there be an exact match between the design and the binding site. If you would like some help with the design, please email geneartsupport@lifetech.com.
Here are common problems, possible causes, and recommendations:
- Smear obtained: Lysate is too concentrated; Dilute lysate 2- to 4-fold and repeat the PCR reaction.
- Disparity in band intensity between amplicons: Lysate concentrations vary between samples; Purify the PCR products with the Invitrogen PureLink PCR Purification Kit. For best results in comparing samples, purify PCR and use the same quantity of DNA in each cleavage assay. 50 ng to 100 ng of DNA is enough for each reaction.
- No PCR product: Poor PCR primer design or GC-rich region; Redesign primers that are 18-22 bp, have 45-60% GC content, and a 52-58°C Tm range. For GC-rich regions, add 1-10 µL of 360 GC Enhancer in a 50 µL reaction and repeat the PCR amplification.
Samples are typically run on an agarose gel, such as an Invitrogen E-Gel EX gel, followed by analysis with image software or by microfluidic electrophoresis.
Yes, our GeneArt Genomic Cleavage Detection Kit can be used to determine the efficiency of nuclease cleavage at a given locus.
We recommend that you resuspend the vector in 50 µL of distilled water or 10 mM Tris-HCl (pH 8.0) and incubate for 1 hour at room temperature. Resuspend the vector DNA by gently pipetting up and down 5-10 times. Store the resuspended DNA at -20°C.
Please email our team, at outlicensing@lifetech.com.
No, we would recommend either lipid-mediated transfection or electroporation. Please review relevant references or consult with the supplier of your cell line for the optimal method of transfection.
Double-stranded DNA breaks can be created at your specified genomic locus by using a pair of Invitrogen GeneArt Precision TAL proteins that have been fused to the Fok1 endonuclease. Using a pair of Precision TAL proteins for the targeting reduces off-target effects. The breaks induced by the Fok1 nuclease domain are subsequently repaired through either of two endogenous cellular mechanisms: nonhomologous end joining (NHEJ), or homology-directed repair (HDR). NHEJ is prone to errors and often introduces a frameshift mutation when it occurs within the coding sequence of a protein-coding gene, effectively silencing the gene. Homologous DNA “donor sequences” can be used with HDR to introduce a defined new DNA sequence. Consequently, a Precision TAL protein fused to a Fok1 endonuclease can be used to induce gene silencing or to accurately insert an engineered DNA fragment into an exact location in the genome.
Truncated TAL Fok1: Recommended for mammalian cells
Native TAL Fok1: Recommended for plants
Native Activators: Higher performance in non-mammalian systems
Truncated MCS: Removing endogenous activator activity
All TALs are expressed as Entry clones (Gateway vector-compatible, flanked by attL1 and attL2).
Fok1 TALs:
- TALs tethered with the Fok1 nuclease may be used for targeting specific genes for silencing.
- Fok1 is a Type IIs restriction endonuclease from Flavobacterium okeanokoites, consisting of an N-terminal DNA-binding domain and a C-terminal nonspecific DNA cleavage domain.
- Fok1 acts as a nuclease pair and binds to the DNA duplex at target sites designated by the binding domains resulting in subsequent cleavage.
VP16 or VP64 TALs:
- Either VP16 or VP64 may be used to increase endogenous or recombinant gene expression levels.
- VP16 is a trans-acting protein originating from the herpes simplex virus, to form a complex with host transcription factors to induce immediate early gene transcription.
- VP64 is a tetrameric form of the VP16 minimal activation domain.
MCS TALs:
- The multiple cloning site (MCS) TAL allows the user to clone any desired effector domain for targeting to any locus within the genome.
Please follow standard plasmid DNA recommendations for transfection. We recommend our Invitrogen Lipofectamine 3000 and Invitrogen Lipofectamine 2000 reagents.
- Design Invitrogen GeneArt TALs for target DNA, and clone into Gateway entry or destination vectors
- In vitro validation of TALs (optional)
- Transfect cells with TAL expression vectors
- TAL-mediated target cleavage
- Cleavage analysis using Invitrogen GeneArt Genomic Cleavage Detection Kit
Yes, KO and KI strains involve editing the native genetic code by either mutating or deleting an encoded message or inserting a new piece of information at a desired site. Although this does manipulate the native genetic information, this technology, when used in a responsible manner, has very useful applications, including engineering yeasts for insulin production or engineering cells for more economically and clinically valuable products.
mRNA and DNA are best delivered via lipid-based transfection for standard test cell lines (i.e., 293, HeLa, etc.). mRNA delivery also reduces the risk of transgene integration. We offer products including our Invitrogen Lipofectamine MessengerMAX Reagent for delivery of mRNA, and Invitrogen Lipofectamine 3000 Reagent for delivery of DNA. For stem cells, electroporation is the best option.
Manufacturing takes place typically within 2 weeks after your order has been received.
By careful designing they can be engineered to be very specific. Recent publications show that 1-3 bp mismatches in target DNA sequences can be tolerated to a large extent.
Yes. If viral-based delivery is your preferred option, we recommend adenoviral systems over lentiviral systems for TAL delivery.
The TAL vector construct is 3.3 kb.
A recent paper (http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2675.html) by Prashant Mali in Nature Biotechnology shows that TALENs are tolerant of 1-2 mismatches, but less tolerant to a large majority of 3 bp mismatches.
We offer the N-TAL Fok1 Entry Gateway vector or the Fok1 CMV promoter–driven vector for expression.
Invitrogen GeneArt PerfectMatch TALs are derived from Invitrogen GeneArt Precision TALs. PerfectMatch TALs have a new design that removes the 5′ base constraint, and therefore, can be designed to target any desired sequence in the genome. They contain 3 amino acids mutated at the N terminus of the TAL effector, which converts the 5′ binding motif to a universal binding motif able to bind to any base: A, G, C, or T. These PerfectMatch TALs can be designed with Fok1 nuclease in a Invitrogen Gateway entry vector or with CMV-driven expression for ready-to-express format for mammalian systems. PerfectMatch TALS perform as well as or better than our original Precision TALs. Currently, PerfectMatch TALs are only available with nuclease function (Fok1 Nuclease Pair), whereas Precision TALs are offered with nuclease function (Fok1 Nuclease Pair), activator function (VP16 or VP64), or custom function (MCS vector).
We compared cleavage efficiencies of PerfectMatch and Precision TALs designed for the HPRT locus using the Invitrogen GeneArt Genomic Cleavage Detection Kit, and found PerfectMatch TALs exhibit cleavage efficiencies equal to or better than the performance of Precision TALs on the same targeted region.
PerfectMatch TALs increase the flexibility of designing TAL effector targets and make it possible to keep the spacing distance between targets of TAL effector pairs at 15–16 bp to get maximal TAL effector efficiency.
Here are our suggestions:
Invitrogen GeneArtPrecision TALs allow the construction of TAL effector functional proteins directed to either 18- or 24-base DNA target sites.
Each target site must be preceded by a 5′ T because the N terminus of the TAL effector protein contains a conserved T-binding motif. The 5′ T does not count as one of the 18 or 24 bases to be selected for targeting your specific site.
Nuclease pairs need to be designed with a spacing of 13-18 bp between the target sites on opposite strands of the DNA.
For Invitrogen GeneArt PerfectMatch TALs, there are no restrictions for the 5′ base. We developed these second-generation TALs by mutating the N-terminal domain to reduce its specificity for 5′ T. Therefore, any 5′ base (T, G, C, or A) can be used with performance comparable to that of the original Precision TALs. We recommend that you design nuclease pairs with a 15-16 bp spacing between the two TAL effectors.
The contribution of individual binding motifs within the DNA-binding domain to TAL effector binding efficiency is thought to differ, since strong and weak binding motifs exist. The A- and T-binding motifs are thought to fall within the “weak binder” category, while the C- and G-binding motifs are thought to be “strong binders”. Stretches of more than 5 weak binders should be avoided at the extreme 5′ end of the binding domain (not counting the 5′ T), or if they are not flanked by Cs.
We recommended that you select a TAL effector with a DNA-binding domain composed of mixed binding motifs for best results. In the context of the living cell, DNA accessibility also determines TAL effector efficiency. It is possible that chromatin, DNA methylation, and/or proteins bound to the DNA may interfere with TAL binding.
Although promoter structure varies, and specific rules regarding design are currently lacking, it is recommended that TAL transcription factors used for transcriptional activation of natural promoters be positioned upstream of the TATA box, or in some cases downstream of the transcriptional start site. Selecting a target site directly over the TATA box or other known transcription factor binding site is not recommended. Be sure that the natural ATG is present, and that no premature ATG which may interfere with the natural translational start is transcribed.
Please view the available effector domains (https://www.thermofisher.com/us/en/home/life-science/cloning/gene-synthesis/geneart-precision-tals.html#table1).
We will ship you a clone with a verified, optimized sequence approximately 2 weeks after confirming your order.
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.
- One-to-one relationship between two critical amino acids in each repeat and each DNA base in the target sequence
- Simple code for creating engineered TALs
- More predictable than zinc fingers
- Modular assembly of domains allows engineering of sequence-specific DNA-binding proteins
- Can be coded to deliver functionality to a specific locus for: nucleases, activators, repressors, chromatin modifiers
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
Genomic DNA at the locus being investigated must be PCR amplified prior to detection. Follow these recommended guidelines to ensure optimal amplification and subsequent detection:
- For best results, use primers with Tm >55°C.
- Design primers that are 18-22 bp in length and have 45-60% GC content.
- For efficient amplification, design primers to yield amplicon lengths between 400 and 500 bp.
- Design primers so that the potential cleavage site is not in the center of the amplicon and that the detection reaction will yield two distinct product bands.
The assay uses genomic DNA extracted from cells transfected with constructs expressing engineered nucleases. Following cleavage, indels are created by the cellular repair mechanisms. Loci where the gene-specific double-stranded breaks occur are amplified by PCR. The PCR product is denatured and reannealed so that mismatches are generated as strands with an indel reannealed to strands with no indel or a different indel. The mismatches are subsequently detected and cleaved by Detection Enzyme, and then the resultant bands are analyzed by gel electrophoresis and band densitometry.
The GeneArt Genomic Cleavage Detection Kit provides a simple, reliable, and rapid method for the detection of locus-specific double-strand break formation. This technique relies on mismatch detection endonucleases to detect genomic insertions or deletions (indels) incorporated during cellular NHEJ repair mechanisms.
GeneArtGenomic Cleavage Selection Kit
- Fast, live detection
- Visual indication (fluorescence)
- Proves editing tool works
- Allows for clone enrichment
GeneArt Genomic Cleavage Detection Kit
- Requires cells to be lysed
- Quantifiable results
- Negative result does not indicate whether editing tool works or not
- No enrichment capabilities
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.
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).
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.