Method for Screening and Identifying Functional lncRNAs

A functional and purposeful technology, applied in the field of screening and identification of functional lncRNAs, can solve problems such as destruction, laborious scale-up, and lack of scale-up

Active Publication Date: 2021-02-19
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • Method for Screening and Identifying Functional lncRNAs
  • Method for Screening and Identifying Functional lncRNAs
  • Method for Screening and Identifying Functional lncRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0096] Materials and methods

[0097] 1. Cells and Reagents

[0098] The HeLa cell line from Z. Jiang's laboratory (Peking University) was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco C11995500BT). The Huh 7.5 cell line from the laboratory of S. Cohen (Stanford University School of Medicine) was cultured in DMEM (Gibco) supplemented with 1% MEM non-essential amino acids (NEAA, Gibco 1140-050). K562 cells from H. Wu laboratory (Peking University) and GM12878 cells from Coriell cell bank were cultured in RPMI1640 medium (Gibco11875-093). All cells were supplemented with 10% fetal bovine serum (FBS, CellMaxBL102-02) and 1% penicillin / streptomycin at 37°C in 5% CO 2 cultivated in.

[0099] 2. Reverse transcription PCR (RT-PCR) to test for intron retention or exon skipping

[0100] Cloning of sgRNA into a lentiviral expression vector carrying a CMV promoter-driven mCherry marker, followed by transduction of HeLa by viral infection at MOIOC cell 1-4 , 72 hours ...

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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] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/86
CPCC12N15/113C12N2310/20C12N2330/31C12N2740/16043C12N9/22
Inventor 魏文胜刘莹曹中正王轶楠郭昱袁鹏飞
Owner PEKING UNIV
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