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Cell:使用CRISPR技术追踪RNA

 包括癌症和自闭症在内的许多疾病都与RNA出现问题有关联,能不能靶向及追踪活细胞中RNA、研究相关的疾病过程呢?别担心,基因编辑工具CRISPR/Cas9可以为我们解决这个问题。

我们知道,CRISPR/Cas9一般识别的是DNA,所以研究人员做了以下改造:
设计了独特的短核酸:PAMmer,使Cas9能在不损伤靶分子的情况下有效识别RNA而非DNA;
利用一种无催化活性的Cas9酶避免了切割转录组,使用荧光蛋白标记Cas9,从而可以在显微镜下监测RNA活动;
优化sgRNA来改善靶向RNA的效率。
上述改造使活细胞中追踪RNA成为可能,关键还不会改变RNA丰度或翻译蛋白的量。
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参阅文献:
Programmable RNA Tracking in Live Cells with CRISPR/Cas9.
Cell. 2016 Mar 16;pii:S0092-8674(16)30204-5. doi:10.1016/j.cell.2016.02.054. 
RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting?methods rely on incorporation of exogenous tags.?Here, we demonstrate that nuclease-inactive S.?pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in?situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in?living cells in a programmable manner without genetically encoded tags.
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