CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.
CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators.
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CIGAR™ seq，一种基于 CRISPR/Cas™ 的方法，用于公正筛选新型 mRNA 修饰调节因子
DOI：
10.15252/msb.202010025
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发表时间：
2020-11
影响因子：
9.9
通讯作者：
Chen W
中科院分区：
文献类型：
--
作者：
Fang L;Wang W;Li G;Zhang L;Li J;Gan D;Yang J;Tang Y;Ding Z;Zhang M;Zhang W;Deng D;Song Z;Zhu Q;Cui H;Hu Y;Chen W
细胞RNA被170多种化学修饰修饰。mRNA中的许多修饰，包括m6 A和m5 C，已经与生理和/或病理条件下的关键细胞功能相关。为了了解这些修饰的生物学功能，确定调节修饰速率的调节剂是至关重要的。然而，到目前为止，还缺乏用于无偏筛选这些调节剂的高通量方法。在这里，我们报告了这样一种方法，将合并的CRISPR筛选和报告基因与RNA修饰读数相结合，称为CRISPR整合的gRNA和报告基因测序（CIGAR-seq）。使用CIGAR-seq，我们发现NSUN 6是一种新的mRNA m5 C甲基转移酶。随后在没有或有NSUN 6和/或NSUN 2敲除的HAP 1细胞中进行的mRNA亚硫酸氢盐测序显示，NSUN 6和NSUN 2作用于mRNA m5 C位点的非重叠子集，并且一起促成了mRNA中几乎所有的m5 C修饰。最后，以m1A为例，我们证明了CIGAR-seq可以很容易地用于识别其他mRNA修饰的调节因子。CIGAR-seq是一种将合并的CRISPR筛选与表位转录组报告基因相结合的新方法，用于鉴定mRNA修饰调节因子。NSUN 6是一种新的mRNA m5 C甲基转移酶，它与NSUN 2一起参与了mRNA中几乎所有的m5 C修饰。
Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high‐throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR‐seq). Using CIGAR‐seq, we discovered NSUN6 as a novel mRNA m5C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non‐overlapping subsets of mRNA m5C sites and together contributed to almost all the m5C modification in mRNA. Finally, using m1A as an example, we demonstrated that CIGAR‐seq can be easily adapted for identifying regulators of other mRNA modification. CIGAR‐seq is a new method combining pooled CRISPR screen with an epitranscriptomic reporter for identifying mRNA modification regulators. NSUN6 is discovered as a novel mRNA m5C methyltransferase, which together with NSUN2, contributes to almost all the m5C modification in mRNA.
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影响因子：
48
作者：
Linder, Bastian;Grozhik, Anya V.;Olarerin-George, Anthony O.;Meydan, Cem;Mason, Christopher E.;Jaffrey, Samie R.
通讯作者：
Jaffrey, Samie R.
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
14.9
作者：
Hauenschild R;Tserovski L;Schmid K;Thüring K;Winz ML;Sharma S;Entian KD;Wacheul L;Lafontaine DL;Anderson J;Alfonzo J;Hildebrandt A;Jäschke A;Motorin Y;Helm M
通讯作者：
Helm M
影响因子：
8.8
作者：
Hussain S;Sajini AA;Blanco S;Dietmann S;Lombard P;Sugimoto Y;Paramor M;Gleeson JG;Odom DT;Ule J;Frye M
通讯作者：
Frye M
影响因子：
4.5
作者：
Edelheit S;Schwartz S;Mumbach MR;Wurtzel O;Sorek R
通讯作者：
Sorek R