1. Academic Validation
  2. BESST: a novel LncRNA knockout strategy with less genome perturbance

BESST: a novel LncRNA knockout strategy with less genome perturbance

  • Nucleic Acids Res. 2023 Mar 20;gkad197. doi: 10.1093/nar/gkad197.
Shikuan Zhang 1 2 3 Yue Chen 4 Kunzhe Dong 5 6 Yiwan Zhao 1 2 3 Yanzhi Wang 1 2 3 Songmao Wang 1 2 3 Chen Qu 1 2 3 7 Naihan Xu 2 3 7 Weidong Xie 2 3 7 Chunyu Zeng 4 Qing Rex Lyu 8 9 Yaou Zhang 2 3 7
Affiliations

Affiliations

  • 1 School of Life Sciences, Tsinghua University, Beijing100084, China.
  • 2 China State Key Laboratory of Chemical Oncogenomics, Tsinghua Shenzhen International Graduate School, Shenzhen518055, China.
  • 3 Key Lab in Healthy Science and Technology of Shenzhen, Tsinghua Shenzhen International Graduate School, Shenzhen518055, China.
  • 4 Department of Cardiology, Daping Hospital, The Third Military Medical University, Chongqing400042, China.
  • 5 Immunology Center of Georgia, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA.
  • 6 Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
  • 7 Open FIESTA Center, Tsinghua University, Shenzhen518055, China.
  • 8 Medical Research Center, Chongqing General Hospital, Chongqing 401147, China.
  • 9 Biomedical and Health Institute, Chongqing Institute of Green and Intelligence Technology, Chinese Academy of Sciences, Chongqing400714, China.
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 ∼130 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.

Figures
Products
Inhibitors & Agonists
Other Products