Genetic tools to dissect functions of long noncoding RNAs

LncRNAs represent an abundant group of noncoding transcripts, some of which carry out important regulatory functions. To survey the biological and molecular roles of lncRNAs, reliable strategies for their genetic inactivation are required. Several lncRNA features make them challenging to target by genome editing. First, lncRNA loci often span large genomic distances. As such, full or partial deletion alleles are not always easy to generate and interpret as they might affect DNA regulatory elements. Second, in contrast to proteins, lncRNA transcripts are usually resistant to the minimally invasive approach of point substitutions. Third, lncRNA sequences exhibit rapid evolutionary turnover, impeding prediction and targeting of the specific functional sequence elements. Nonetheless, advances in genome editing and comparative genomics have expanded the repertoire of genetic strategies to dissect lncRNA functions in model organisms and cell lines. In this review, we discuss several approaches that have been used to generate lncRNA mutant alleles, focusing on vertebrate lncRNAs. We also briefly highlight comparative genomics approaches to identify conserved lncRNA sequence motifs, which represent attractive target sequences to abrogate lncRNA functions and to pinpoint functional contributions of these elements.