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.

中文翻译：

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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


细胞 RNA 被 170 多种化学修饰修饰。 mRNA 的许多修饰（包括 m ⁶ A 和 m ⁵ C）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因子至关重要。然而，迄今为止缺乏对这些调节剂进行公正筛选的高通量方法。在这里，我们报告了一种将 CRISPR 筛选和报告基因与 RNA 修饰读数相结合的方法，称为C RISPR整合g RNA和报告基因测序 (CIGAR-seq)。使用 CIGAR-seq，我们发现 NSUN6 作为一种新型 mRNA m ⁵ C 甲基转移酶。随后在没有或有 NSUN6 和/或 NSUN2 敲除的 HAP1 细胞中进行的 mRNA 亚硫酸氢盐测序表明，NSUN6 和 NSUN2 对 mRNA m ⁵ C 位点的非重叠子集起作用，并共同促成了 mRNA 中几乎所有的 m ⁵ C 修饰。最后，以 m ¹ A 为例，我们证明了 CIGAR-seq 可以轻松用于识别其他 mRNA 修饰的调节因子。