Genome oligopaint via local denaturation fluorescence in situ hybridization.
Genome oligopaint via local denaturation fluorescence in situ hybridization.
复制标题
局部变性荧光原位杂交技术检测基因组寡核苷酸。
DOI：
10.1016/j.molcel.2021.02.011
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发表时间：
2021-04-01
期刊：
影响因子：
16
通讯作者：
Ha T
中科院分区：
文献类型：
--
作者：
Wang Y;Cottle WT;Wang H;Feng XA;Mallon J;Gavrilov M;Bailey S;Ha T
Cas9与可编程指导RNA复合靶向特异性双链DNA进行切割。通过利用Cas9作为超解旋酶到基因组DNA的可编程加载器，在这里，我们报告了一种生理温度DNA FISH方法，称为通过局部变性荧光原位杂交的基因组寡聚物（GOLD FISH）。代替常规DNA FISH中的全局变性，在Cas9产生的切口处加载超解旋酶允许局部DNA变性，减少探针的非特异性结合并避免苛刻处理如热变性。GOLD FISH依赖于Cas9切割靶DNA序列，并避免了与依赖于Cas9结合的其他基因组标记方法相关的高核背景。出色的信号亮度和特异性使我们能够对非重复基因组DNA位点进行成像，并分析活性和非活性X染色体之间的构象差异。最后，GOLD FISH可用于快速鉴定患者组织中的HER2基因扩增。Wang等人开发了通过局部变性荧光原位杂交（GOLD FISH）的基因组寡聚蛋白，这是一种利用Cas9和超解旋酶使靶DNA局部变性以用于探针结合的DNA FISH方法。GOLD FISH避免了热变性等苛刻条件，并以高信号背景比稳健地检测非重复DNA基因座。
Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, here we report a physiological-temperature DNA FISH method termed Genome Oligopaint via Local Denaturation Fluorescence in Situ Hybridization (GOLD FISH). Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing non-specific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image non-repetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in a patient tissue. Wang et al. develop Genome Oligopaint via Local Denaturation Fluorescence in Situ Hybridization (GOLD FISH), a DNA FISH method utilizing Cas9 and superhelicase to locally denature target DNA for probe binding. GOLD FISH avoids harsh conditions such as heat denaturation and robustly detects non-repetitive DNA loci with high signal-to-background ratio.
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