版权所有:http://epigenie.com/products/enchip-kit/
Catalog No.53125.
什么是enChIP:
Chromatin Immunoprecipitation (ChIP) 染色体免疫沉淀,是用来研究蛋白质与DNA直接相互作用的方法。蛋白质包括转录因子,系统调节蛋白,修饰的组蛋白,染色质修饰蛋白酶和聚合酶。原因是这个技术可以知道这些蛋白结合在特定的位置。engineered DNA-binding molecule-mediated ChIP(enChIP)在此基础上利用了CRISPR/Cas9技术来研究染色质的相互作用。
可以实现分离特定基因组位置的ChIP-seq。虽然从结构上看比起ChIP-seq的全基因组范围范畴上见效了,但是定点ChIP对于生物化学研究还是很重要的。
原始文章可以点下面链接:
https://www.sciencedirect.com/science/article/pii/S0006291X13013296
CRISPR (clustered regularly interspaced short palindromic repeats)系统用来指导guide RNA (gRNA)到指定的基因组位置,来进行免疫沉淀反应。值得注意的是,目前大概有三种不同的CRISPR/Cas system。区别在于他们采用不同的Cas蛋白质。其中Type II system使用的是Cas9蛋白结合体。也是目前最热门的CRISPR/Cas9 system。
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介绍 enChIP
原文: http://www.activemotif.com/catalog/1172/enchip
Chromatin Immunoprecipitation (ChIP) 是一种有效的方法来研究蛋白质/DNA之间的相互作用。其中包括转录因子(transcription factors),协同蛋白(co-regulatory proteins),组蛋白修饰,染色体修饰蛋白以及聚合酶。因为ChIP能够识别蛋白质特异性结合在DNA序列的位置。利用CRISPR/Cas9技术,新型的应用可以用于研究染色体的相互作用。
enChIP,全称是engineered DNA-binding molecule-mediated chromatin immunoprecipitation,译为使用设计的能结合DNA的特异分子的组蛋白免疫沉淀。CRISPR系统被用于携带gRNA(guideRNA)到指定的基因组位置。gRNA是自己设计的而且与对应的序列是互补的。gRNA和Cas9是同时表达,这里用到的Cas9是酶处理过的,不会剪切序列,叫做dCas9 (deactivated Cas9)。dCas9蛋白是由Active Motif公司的特殊含有AM-tag序列表达出来,是专门设计给ChIP用的。AM-tag的在哺乳动中没有类似的蛋白,而且结构上不复杂,能够很大程度上让AM-tag暴露在外面,使得抗体能够识别。
Following transfection of the gRNA and AM-tagged dCas9, cells are formaldehyde fixed, lysed and the chromatin is fragmented by sonication.
转染gRNA和AM-tagged dCas9之后,细胞会用福尔马林固定,再进行细胞裂解,染色质会由超声波进行切段。
An antibody directed against the AM-tag is used to enrich for genomic sequences bound by the gRNA/dCas9 complex.
可以识别AM-tag的抗体用来富集绑有gRNA/dCas9的DNA序列。
Following immunoprecipitation, DNA can be analyzed by qPCR or NGS to identify the enriched genomic regions.
在进行免疫沉淀以后,DNA序列可以用qPCR或者次世代测序来识别富集的基因组序列。
enChIP provides the opportunity to investigate off-target effects of gRNA design, or to study cis- and trans-interacting chromosomal looping events.
enChIP使得调查gRNA脱靶提供了机会,而且也可以用于研究启动子以及转录因子在内的一些染色体环状结构是否发生。
To use Active Motif’s enChIP® Kit simply clone your gRNA sequence into one of Active Motif’s expression vectors.
如果使用AM公司的enChIP试剂盒,要先简单的克隆你设计的gRNA序列到AM的表达载体质粒。
Following transfection and expression of the gRNA and AM-tagged dCas9, the enChIP Kit can be used to isolate chromatin and perform immunoprecipitation using an antibody directed against the AM-tag.
在转染和表达AM-tagged dCas9之后,enChIP试剂盒可以用来分离染色体并且进行免疫沉淀,免疫沉淀使用特异识别AM-tag的抗体。
The enChIP Kit contains sufficient reagents to perform 16 chromatin preparation and immunoprecipitation reactions. enChIP试剂盒足以满足准备16次染色体提取和免疫沉淀反应所需的试剂量。
The CRISPR/Cas system can be traced to bacteria and archaea which use the system to target and inactivate foreign genetic material.
CRISPR/Cas系统可以追溯到细菌和微生物,它们用于目标和抑制外来遗传物质。
This adaptive immunity mechanism relies on the acquisition of spacers, the assembly of the CRISPR RNAs (crRNAs) and Cas proteins to the invading nucleic acids and degradation of the foreign material by nucleases.
这一适应性免疫机制依靠CRISPR RNA以及能识别入侵核酸的Cas蛋白并且能够让核酸酶去降解外来物质。
There are three main types of CRISPR/ Cas systems, each with a specific Cas protein. Type II systems utilize Cas9 complexes.
主要有三种型号的CRISPR/Cas系统,主要是根据不同的Cas蛋白。II型系统是依靠Cas9蛋白结构。
Recently, researchers have repurposed the CRISPR/Cas9 system for genome engineering of mammalian systems.
目前,研究者已经利用CRISPR/Cas9系统来设计哺乳类动物的基因组。
In type II CRISPR/Cas systems, the crRNA hybridizes with a transactivating crRNA (tracrRNA) that contains additional nucleotide sequences.
在II型CRISPR/Cas系统中,CRISPR RNA与带有多余核酸序列的返式RNA结构互补结合。
In the presence of Cas9, the crRNA-tracrRNA hybrid is processed to generate a spacer that is 20 nucleotides in length.
在有Cas9存在,crRNA-tracrRNA会产生一个20个核苷酸长度的spacer。
The Cas9 complex then undergoes a conformational change for DNA binding.
Cas9结构然后会经历构象结构的改变为了与DNA序列结合。
The spacer sequence directs the Cas9 complex to a complementary DNA sequence containing a protospacer adjacent motif, or PAM site (5 ́-NGG).
spacer序列能够指导Cas9结构去往特定DNA序列(包含protospacer adjacent motif, or PAM site (5 ́-NGG))。
Recognition of a PAM site leads to unwinding of the DNA and formation of an RNA-DNA heteroduplex. Nucleases are then activated to create a double-stranded break in the target DNA.
当识别PAM区域以后,会促使DNA解旋,RAN会与DNA产生异源双链核酸分子。进而核酸被激活产生一个断开的双链DNA结构。
Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) was first described in 2013 by Toshitsugu Fujita and Hodaka Fujii.
enChIP是2013年日本的Fujita与Fujii在他们发表的文章里首先提到的。
enChIP was designed to purify specific genomic regions using the CRISPR/Cas9 system.
enChIP被设计为纯化特殊的基因组区域,通过使用CRISPR/Cas9系统。
A single guide RNA vector containing a fusion of the crRNA-tracrRNA sequence (5 ́side to identify the DNA target sequence and a 3 ́duplex RNA structure to bind Cas9) is used to clone the target sequence of interest.
单一的指导RNA载体包含一个融合物,crRNA-tracrRNA序列(5‘端用于识别DNA靶向序列,3’端是重复的RNA结构,用来结合Cas9)用以克隆感兴趣的序列。
When the gRNA is expressed in combination with a tagged deactivated Streptococcus pyogenes Cas9 (dCas9) protein, specific genomic regions can be immunoprecipitated using an antibody directed against the tag.
当gRNA表达并且与dCas9蛋白结合,接下来特定的靶向基因组区域就可以被tag-antibody免疫沉淀下来。
By using a catalytically inactive form of Cas9 endonuclease, double-stranded breaks are not introduced and DNA, RNA and proteins associated with the target sequence can be recovered.
由于采用的Cas9是催化失活的,所以不会产生双链断开,所以和那段序列结合DNA,RNA以及蛋白都可以得到 (fig 1)。
With Active Motif’s enChIP Kit, simply clone your 20 nucleotide target sequence into Active Motif’s gRNA or dCas9 expression vectors.
使用AM的enChIP试剂盒,简单的克隆你的20个核苷酸的靶向核苷酸序列到AM的gRNA或dCas9表达载体里面。
For researchers looking to perform genome editing experiments, determining the specificity of the gRNA binding site is critical.
研究者如果打算进行基因组改变实验,识别gRNA结合的特定序列会非常严格。
If the gRNA target sequence demonstrates off-target binding, then genome editing methods using active Cas9 protein could lead to unwanted downstream effects.
如果gRNA靶向的序列被证实脱靶集合,采用Cas9基因组修饰的方法会导致未来结果不可预测。
By using the enChIP Kit containing the dCas9 protein, off-target gRNA binding sites can be identified to determine the quality of the gRNA design prior to use in genome editing experiments.
使用dCas9蛋白如果出现脱靶现象会可以在执行基因组编辑之间鉴定gRNA的质量。
Additionally, enChIP can be used to study cis- and trans- chromosomal interactions.
另外,enChIP可以用来研究顺势,转势的一些染色体相互作用。
Evaluation of the immunoprecipitated DNA sequences may reveal chromosomal looping events.
通过评估沉淀的DNA序列,就可以揭示一些染色体环状结构。
如何设计gRNA
CRISPRdirect - http://crispr.dbcls.jp
Michael Boutros Lab - http://www.e-crisp.org/E-CRISP/designcrispr.html
Feng Zhang Lab - http://crispr.mit.edu
以上是in vivo的enChIP,同时还有一个in vitro enChIP
实验方法请参考:
https://bio-protocol.org/e2612