Table 1.

Advantages and disadvantages of the different RNAi-based silencing systems

RNAi molecule	Activity	Advantage	Disadvantage
MicroRNAs [32]	Naturally occurring small RNA molecule responsible for regulation of mRNA translation	Naturally occurring, some are known to be involved in cancer and virus regulation [43–45] Active in the cytoplasm Enters RISC directly	Highly unstable in physiological conditions May have multiple mRNA targets, resulting in off-target side effects
Synthetic small interfering RNA (siRNA) [27]	Synthetic double-stranded RNA molecule with complementary sequence to target gene	Specifically designed to target the gene of interest, minimising off-target effects Highly effective in laboratory environments Active in the cytoplasm Enters RISC directly	Highly unstable in physiological conditions May activate an immune response
Short hairpin RNA (shRNA) [21]	Artificial RNA molecule with an artificially designed tight hairpin loop. One stem sequence is complementary to the target gene	Can be delivered as a plasmid or virus vector with a poll III promoter for more stable and longer-lasting treatment Enters the RNAi pathway at an earlier step than siRNA, which may be advantageous for activity	Needs to be delivered to the nucleus, requiring crossing of the nuclear membrane May activate an immune response May result in excess RNAi molecule expression, leading to saturation of the natural miRNA pathways and resulting in severe toxicities [46]
Mirised short hairpin RNA (shRNAmir) [39, 40]	Artificial RNA molecule with a hairpin loop and bulges derived from a native microRNA. One stem sequence is complementary to the target gene	More stable as delivered via a plasmid or virus vector Mimics the native miRNAs more closely, therefore recognised by the RNAi machinery more accurately Enters the RNAi pathway at an earlier step than siRNA	Needs to be delivered to the nucleus, requiring crossing of the nuclear membrane May activate an immune response May result in excess RNAi molecule expression, leading to saturation of the natural miRNA pathways and resulting in severe toxicities [46]
Artificial MicroRNA cluster [42]	Artificial RNA molecule with multiple stem loops derived from a native microRNA cluster, i.e. mir-17-92, stem sequences have been replaced to be complementary to the target gene/s	As above, with the additional benefit of delivering multiple RNAi molecules for targeting of several sites of one gene or different genes Avoidance of escape mutants	As above First RNAi molecule is expressed at higher levels than remaining molecules [47]