Fig. 1

Reverse-genetics approaches for lncRNA functional studies. The illustration shows various methods that target either RNA (based on RNAi, ASOs, or CRISPR/Cas13) or DNA, based on the CRISPR/Cas9 family of methods that can cause deletions and insertions of specific sequences (e.g., polyA cassettes or self-cleaving ribozymes) or bring transcription activators/silencers to promoters depending on specific system employed. Also shown are some of the known problems with these techniques—off-target effects caused by partial sequence matches (1, 4, 6) or non-specific effects such as triggering innate immune response (2), saturation of the endogenous RNAi machinery (3), and interactions with proteins (5), as well inability to discriminate between the targets and other overlapping (7) or shared elements (8) and to target sequences containing repetitive elements (9). More details are in the text