CUT&RUN（Cleavage Under Targets and Release Using Nuclease）是一項用于在細(xì)胞天然染色質(zhì)環(huán)境下檢測蛋白質(zhì)-DNA相互作用的強(qiáng)大技術(shù)。在CUT&RUN中，蛋白A和蛋白G與微球菌核酸酶(pAG-MNase)融合，并選擇性地切割抗體標(biāo)記的染色質(zhì)，經(jīng)過剪切后的片段從細(xì)胞中分離出來，純化，最后通過NGS進(jìn)行分析。作為ChIP的高效替代方案，CUT&RUN克服了傳統(tǒng)ChIP-seq分析法的許多缺點(diǎn)。

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與ChIP-seq相比CUT&RUN的優(yōu)勢?

● 需要的細(xì)胞數(shù)量較少：CUTANA? CUT&RUN分析只需要5,000個細(xì)胞即可生成高分辨率的結(jié)果。

●?操作步驟簡單：CUTANA? CUT&RUN在3天內(nèi)即可完成從細(xì)胞到文庫的建立。還適用于多道移液器和8聯(lián)排管，提高了分析的重復(fù)性和通量。

●?測序成本降低：只需要300 - 800萬個測序讀段，高通量測序可以檢測更多樣本。

●?減少實(shí)驗(yàn)中需要優(yōu)化的步驟：CUT&RUN跳過了ChIP-seq中最具挑戰(zhàn)性的部分(包括交聯(lián)、染色質(zhì)片段化和免疫沉淀(IP))，只需要較少的優(yōu)化步驟。

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EpiCypher可提供包括CUTANA?CUT&RUN Assay Kit和 Library Prep?Kit，以及一系列不斷擴(kuò)大的經(jīng)過CUT&RUN驗(yàn)證的抗體產(chǎn)品。SNAP-CUTANA??K-MetStat?Panel提供了一個基本的檢測控制，可用于抗體驗(yàn)證，實(shí)驗(yàn)流程的優(yōu)化，并作為實(shí)驗(yàn)成功與否的直接衡量標(biāo)準(zhǔn)。下面重點(diǎn)介紹了一些研究，展示了CUTANA CUT&RUN在不同的研究領(lǐng)域的應(yīng)用。希望這些文獻(xiàn)能夠?yàn)镃UT&RUN如何應(yīng)用于您的項目提供思路。

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1.?Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer–promoter interactions

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Wang et al. Nucleic Acids Research, 2022.?PMID:?35849129

細(xì)胞類型：Transfected HeLa cells and HEK293T cells

目標(biāo)蛋白：H3K27ac

主要內(nèi)容：argeted modification of the epigenome represents a promising strategy for precision medicine, since it allows activation of genes without disrupting DNA sequence. CRISPR/Cas9 has been?cleverly modified for this purpose?by fusing a nuclease-defective Cas9 (dCas9) with transcriptional activation domains, such as histone lysine acetyltransferases CBP or p300. Here, Wang et al. used CUT&RUN to help analyze local and genome-wide changes in chromatin structure when using different dCas9 activation systems. In agreement with previous studies, they found that?dCas9 activators?were highly variable, with results varying by the type of dCas9 fusion protein, cell type, and genomic target. They also discovered that targeting dCas9 activators to enhancers can induce reciprocal epigenomic changes at target promoters, leading to increased gene expression.

上榜理由：雖然dCas9激活系統(tǒng)提供了特定的基因激活，但表觀基因組變化也是可能的。CUT&RUN做為一種快速可靠的染色質(zhì)分析檢測方法，可幫助分析使用不同dCas9激活系統(tǒng)時，染色質(zhì)結(jié)構(gòu)的局部和全基因組變化。

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2.?Histone H3 proline 16 hydroxylation regulates mammalian gene expression

Liu et al. Nature Genetics, 2022.?PMID:?36347944

細(xì)胞類型：MDA-MB-231 cells (hypoxia-sensitive breast cancer cells) and 293T cells; includes experiments with and without knockdown of EGLN2 using the inducible CRISPR v2 system

目標(biāo)蛋白：H3P16oh, EGLN2, KDM5A, H3K4me3

主要內(nèi)容：Low oxygen (hypoxia)?induces transcriptional changes that drive tumor growth and increase cancer severity. Although multiple studies show that?chromatin?is?responsive to hypoxia?and plays a role in these processes, additional information is needed to provide a cohesive mechanism. Here, Liu et al. defined a novel histone PTM directly linked to hypoxia: prolyl (proline) hydroxylation on histone H3, proline 16. As part of this work, the authors used CUTANA CUT&RUN to validate H3P16oh antibodies, characterize its enrichment, and determine its function during hypoxia. Their experiments revealed substantial crosstalk between H3P16oh and H3K4me3, establishing H3P16oh as a hypoxia-sensitive regulator of gene expression and cell proliferation in breast cancer cell lines.

上榜理由：這篇文章為如何使用CUTANA CUT&RUN分析法來研究新的PTMs提供了重要思路。Li等人還展示了組蛋白PTMs如何調(diào)節(jié)疾病中的染色質(zhì)結(jié)構(gòu)和基因表達(dá)，為表觀遺傳學(xué)靶向藥物開發(fā)提供了支持。

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3.?Mapping?cis-regulatory elements in human neurons links psychiatric disease heritability and activity-regulated transcriptional programs

Sanchez-Priego et al. Cell Reports, 2022.?PMID:?35649373

細(xì)胞類型：?Excitatory glutamatergic neurons and inhibitory GABAergic neurons derived from human pluripotent stem cells, with and without stimulation (membrane depolarization)

目標(biāo)蛋白：H3K27ac (0, 30, 90 min), FOS (0, 2 hr)

主要內(nèi)容：Genetic risk variants for psychiatric diseases are?concentrated in?cis-regulatory DNA, suggesting roles in cell-type specific gene expression. However, due to the inherent?challenges?of studying human brain tissues, these genomic regions remain largely unexplored. In this paper, Sanchez-Priego et al. generated large amounts of excitatory and inhibitory neurons using human pluripotent stem cells. They profiled cells using a variety of techniques, including CUT&RUN, ATAC-seq, and RNA-seq, to identify putative?cis-regulatory elements (i.e. enhancers) associated with?activity-dependent gene?expression. To support the relevance of their results to human disease, the authors compared the list of candidate enhancers to a large database of psychiatric-disease risk variants, which revealed significant links to schizophrenia, ADHD, and bipolar disorder.

上榜理由：Sanchez-Priego等人沒有只針對個體變異進(jìn)行研究，而是采取了整體法，他們使用多種技術(shù)，包括CUT&RUN, ATAC-seq和RNA-seq對細(xì)胞進(jìn)行分析，定義疾病相關(guān)細(xì)胞類型中的表觀基因組元素，并與不同數(shù)據(jù)庫進(jìn)行比較引用。

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4.?Acute depletion of human core nucleoporin reveals direct roles in transcription control but dispensability for 3D genome organization

Zhu et al. Cell Reports, 2022.?PMID:?36323253

細(xì)胞類型：HCT116 cell line (all targets), HeLa cell line (NUP93 only)

目標(biāo)蛋白：NUP93, NUP35, NUP205, BRD4, SEC13

主要內(nèi)容：The?nuclear pore complex?acts as the gateway to the nucleus in eukaryotic cells and is composed of ~30 different nucleoporin proteins (NUPs). There is?ample evidence?that the nuclear pore complex helps regulate?3D chromatin organization?and?transcription. However, the functions of NUP subunits in human cells are not defined. Here, Zhu et al. studied NUPs using multiple techniques, including CUTANA CUT&RUN, Hi-C, PRO-seq, and CRISPR/dCas9 tethering. CUT&RUN showed that NUP proteins were generally bound to active chromatin regions. NUP93 specifically associated with promoters and enhancers and directly regulated transcription (similar to?Brown et al.?and?Ibarra et al.). Strikingly, the authors found that core NUP proteins were not required for 3D chromatin architecture, in contrast to?leading hypotheses?in the field.

上榜理由：CUTANA CUT&RUN的一個關(guān)鍵優(yōu)勢是能夠在自然條件下定位大分子復(fù)合物的亞基，而不需要交聯(lián)。值得注意的是，在哺乳動物細(xì)胞中研究NUP蛋白和核孔復(fù)合物一直具有挑戰(zhàn)性，而利用CUT&RUN分析技術(shù)更能夠快速的助力這項研究。

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