CRISPR-RfxCas13d/BSJ-gRNA大规模功能性环状RNA筛选质粒载体-BioVector NTCC Inc.

BioVector® CRISPR-RfxCas13d/BSJ-gRNA大规模功能性环状RNA筛选质粒载体

BioVector® CRISPR–RfxCas13d，通过gRNA靶向circRNA的反向剪接位点（BSJ），可有效区分circRNA与线性mRNA

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 BioVector® 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.

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