State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Nat Protoc. 2022 Sep;17(9):2085-2107. doi: 10.1038/s41596-022-00715-5. Epub 2022 Jul 13.
Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3-4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.
环状 RNA（circRNAs）是由前体 mRNA 外显子反向剪接产生的共价封闭的单链 RNA，最近它们作为基因表达调控中的一类重要分子而出现。circRNAs 与它们的同源线性 mRNA 共享重叠序列，除了反向剪接连接（BSJ）位点。这个特点使得区分 circRNAs 和它们的同源 mRNA 的功能变得困难。我们之前报道了可编程的 RNA 引导的、靶向 RNA 的 CRISPR-Cas13（RfxCas13d）系统可以通过使用靶向 BSJ 位点序列的向导 RNA（gRNA）有效地、特异性地将 circRNAs 与 mRNAs 区分开来。在这里，我们描述了一个基于这个 RfxCas13d/BSJ-gRNA 系统的详细方案，用于在人类细胞系中进行大规模的功能性 circRNA 筛选。该方案包括 gRNA 文库的设计、构建和转导，筛选结果的分析以及功能性 circRNA 候选物的验证。总的来说，这个方案需要一个经过良好训练的分子生物学家和一个生物信息学专家之间大约 3-4 个月的合作。这个方案可以应用于细胞和体内，以识别影响细胞生长的高表达 circRNAs，无论是在未受干扰的条件下还是在环境刺激下，而不会干扰它们的同源线性 mRNA。
