小柯机器人

中国科学院分子细胞科学卓越创新中心陈玲玲等研究人员合作使用CRISPR–Cas13系统对功能性环状RNA进行筛选。这一研究成果于2020年12月7日在线发表在国际学术期刊《自然—方法学》上。

研究人员发现，CRISPR–RfxCas13d可通过使用向导RNA的靶向序列来有效地区分环状RNA（circRNA）与mRNA。使用靶向高表达人类circRNA的反向剪接连接（BSJ）位点之间序列的慢病毒文库，研究人员发现一组circRNA对细胞生长非常重要，主要以细胞类型特异性方式发生，并且常见的致癌性circRNA（circFAM120A）通过阻止序列相似性120A家族的mRNA（FAM120A）结合翻译抑制因子IGF2BP2来促进细胞增殖。

RfxCas13d–BSJ-gRNA筛选的进一步应用发现了circMan1a2，它在小鼠胚胎着床前的发育中具有调节潜力。总之，这些结果将CRISPR–RfxCas13d确立为在单个和大规模水平上发现circRNA并对其功能进行研究的有用工具。

据介绍，由反向剪接外显子产生的circRNA广泛表达，但是由于缺乏区分circRNA与重叠外显子的同源mRNA的适当方法，单个circRNA的功能仍然知之甚少。

附：英文原文

Title: Screening for functional circular RNAs using the CRISPR–Cas13 system

Author: Siqi Li, Xiang Li, Wei Xue, Lin Zhang, Liang-Zhong Yang, Shi-Meng Cao, Yun-Ni Lei, Chu-Xiao Liu, Si-Kun Guo, Lin Shan, Man Wu, Xiao Tao, Jia-Lin Zhang, Xiang Gao, Jun Zhang, Jia Wei, Jinsong Li, Li Yang, Ling-Ling Chen

Issue&Volume: 2020-12-07

Abstract: Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR–RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d–BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR–RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

DOI: 10.1038/s41592-020-01011-4

Source: https://www.nature.com/articles/s41592-020-01011-4

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex