BESST: a novel LncRNA knockout strategy with less genome perturbance.
Nucleic Acids Res
; 51(9): e49, 2023 05 22.
Article
in En
| MEDLINE
| ID: mdl-36938886
ABSTRACT
Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of â¼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.
Full text:
1
Database:
MEDLINE
Main subject:
Genome
/
Genomics
/
Gene Knockout Techniques
/
RNA, Long Noncoding
Limits:
Animals
Language:
En
Journal:
Nucleic Acids Res
Year:
2023
Type:
Article
Affiliation country:
China