Functional knockout of long non-coding RNAs with genome editing

Abstract

An effective loss-of-function study is necessary to investigate the biological function of long non-coding RNA (lncRNA). Various approaches are available, including RNA silencing, antisense oligos, and CRISPR-based genome editing. CRISPR-based genome editing is the most widely used for inactivating lncRNA function at the genomic level. Knocking out the lncRNA function can be achieved by removing the promoter and the first exon (PE1), introducing pre-termination poly(A) signals, or deleting the entire locus, unlike frameshift strategies used for messenger RNA (mRNA). However, the intricate genomic interplay between lncRNA and neighbor genes makes it challenging to interpret lncRNA function accurately. This article discusses the advantages and disadvantages of each lncRNA knockout method and envisions the potential future directions to facilitate lncRNA functional study.

LncRNAway: a web-based sgRNA design tool for precise and effective suppression of long noncoding RNAs

“…While these tools adequately facilitate the design of sgRNAs targeting protein-coding genes, they may not fully address the requirements for studying functional knockouts of lncRNAs. As previously discussed, achieving an effective lncRNA knockout entails considering additional factors due to the distinct features of lncRNAs compared to mRNAs ( 17 ). The launch of LncRNAway will help scientists, especially those focused on lncRNA function study, by providing a streamlined lncRNA knockout/knockdown guidance.…”

“…When considering lncRNA functional knockout, scientists seek high repression efficiency of target lncRNA transcripts, less non-specific impact on neighboring genes resulting from genome manipulation, minimal disturbance to genomic DNA, and ease of execution. In our previous study, we introduced a novel lncRNA knockout strategy by removing the branch point to the 3’ splicing site of the last intron of target lncRNA (BESST), achieving exceptional knockout efficiency and specificity comparing to the common-used promoter-exon 1 (PE1) deletion methods ( 17 , 18 ). The BESST approach can achieve an average of 72% efficiency in repressing lncRNA by removing as few as ∼37 bp from genomic DNA ( 18 ).…”