Method for Screening and Identifying Functional lncRNAs

Abstract

Provided is a high-throughput method for screening or identifying long non-coding RNAs by CRISPR system, which uses paired guide RNA targeting the genomic sequence within the region spanning -50 bp to+75 bp surrounding a splice donor site or a splice acceptor site of a long non-coding RNA.

Description

[0001] field of invention [0002] The present invention relates to screening and identification of functional lncRNAs by performing gene interference on long non-coding RNAs (lncRNAs) by targeting splicing sites in eukaryotic cell genomes. [0003] Background of the invention [0004] As a powerful genome editing tool, the CRISPR-Cas9 system has been used to identify gene function through large-scale screening 1-4 . Even at the genome scale, gene interference is mostly achieved through frameshift mutations generated within exons. In addition to about 2% of protein-coding genes in the human genome, there is more evidence that a large number of remaining transcripts are non-coding RNAs 5 . Among them, lncRNAs >200 nucleotides represent a large subgroup of genes with no apparent protein-coding potential 6-7 . Previous studies have shown that the total number of human lncRNAs exceeds the total number of protein-coding genes and the number continues to climb 8 . [0005] ...

AI Technical Summary

Problems solved by technology

Although tens of thousands of loci in the human genome have been annotated as encoding long noncoding RNAs (lncRNAs), their functions are largely unknown, mainly due to the lack of scalable methods for inducing loss of function of such genes

In general, since lncRNAs are not sensitive to reading frame changes, it is difficult to disrupt their expression in a routine way using the CRISPR-Cas9 system, let alone at a large scale.

We previously developed a deletion strategy via pgRNA library for loss-of-function screening of lncRNAs 9 , but its scaling is laborious

Although studies have demonstrated that based on RNA interference 10,11 or CRISPRi 12 Screening is effective for identifying lncRNA functions, but RNAi approaches have potential off-target issues 13 , and both approaches are limited by the effectiveness of transcriptional knockdown

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