siRNA (Small Interfering RNA)

RNA interference (RNAi) is triggered by the endogenously present single-stranded hairpin microRNAs (miRNA) in cells, small interfering RNAs (siRNA), or short hairpin RNAs (shRNA). RNA interference is a double stranded RNA mediated homology based mechanism evolved to post-transcriptionally regulate eukaryotic gene expression and to serve in host immunity. Although RNAi was adopted as a gene silencing technique first, CRISPR has surpassed RNAi in popularity due to several advantages, for example, its specific yet versatile nature, made possible by advancements that have refined CRISPR technology. A large array of libraries are now available to specifically target many genes in several organisms. At the same time, the choice remains with the user to perform knockouts, knock-ins, or knockdown experiments, making CRISPR extremely versatile.

What is siRNA (Small Interfering RNA)?

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length, and is able to regulate the expression of genes, by a phenomenon known as RNAi (RNA interference). It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. siRNA can be used as tools to study single gene function both in vivo and in vitro. siRNA has also gained extensive attention as a potential therapeutic reagent due to its ability to inhibit specific genes in many genetic diseases, especially against undruggable targets for the treatment of cancer and other diseases. For any new therapeutics, safety is still the primary concern. While the off-target effect of siRNA is a major issue that needs to be addressed by improving the knowledge in this area.

Mechanism of siRNA (Small Interfering RNA)

The core operation of the siRNA involves two steps. The first step of siRNA involves processing and cleavage of longer double-stranded RNA (dsRNA) by an RNAse III type endoribonuclease called Dicer to generate ~21 nucleotide long siRNAs, generally bearing a 2 nucleotide overhang on the 3′ end of each strand, and a 5' phosphate group. Dicer is a multi-domain RNase III-related endonuclease responsible for processing dsRNA to mature siRNA, and transfers the processed products to the RNA induced silencing complex (RISC). When formed, siRNA bound Dicer will transfer small dsRNA to Argonaute, with the help of double-stranded Tat-RNA-binding protein (TRBP) or PACT (PKR activating protein) to mediate RNA interference. This whole protein-RNA complex is called RISC. Human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA.

Schematic of the siRNA mediated RNA interference pathway

Fig 1. Schematic of the siRNA mediated RNA interference pathway.

Duplex siRNA in association with holo-RISC, composed of at least Ago-2, Dicer and TRBP, is identified as the RISC loading complex (RLC). In the RLC, siRNA strands are separated, resulting in the departure of the passenger strand. The antisense single-stranded siRNA component then guides and aligns the RISC complex on the target mRNA. Upon engagement with a long homologous RNA target that pairs with the guide RNA, Argonaute (Ago2) will be activated and degrade the target RNA though its RNAse-H like activity. This is referred to as the cleavage-dependent pathway. There is also a cleavage-independent by-pass pathway, in which the passenger strand with mismatches is induced to unwind and depart by an ATP dependent helicase activity. The RISC with single-stranded guide strand siRNA is then able to execute multiple rounds of RNA interference. ATP is not required for shRNA processing, RISC assembly, cleavage-dependent pathway, or multiple rounds of target-RNA cleavage.

Human Dicer is an integral component of the RNA interference pathway. While, Dicer knockout ES cells can effectively load processed siRNA onto RISC and carry out RNA interference as efficiently as Dicer + ES cells. It appears that in mammalian cells, a perfectly processed siRNA can be effectively loaded onto RISC for RNAi without the help of the TRBP/PACT/Dicer complex. The TRBP/PACT/Dicer complex, however, is required to process either shRNA or long dsRNA to appropriate size and form for their loading onto RISC. This is consistent with previous research that Ago1 and Ago2 containing RISC were found both in the cytoplasm and nucleus, and the nuclear RISC (nRISC) is a complex that is 20× smaller in size than the cytoplasmic RISC (cRISC). RNAi could also be induced by direct exogenous supply of short double stranded RNAs that are ~21 nucleotides in length, called small interfering RNAs (siRNA).

Comparation of siRNA and CRISPR

Techniques such as CRISPR and RNAi are used to modify genes with high precision. CRISPR is a naturally occurring prokaryotic immune defense mechanism that has been recently used for eukaryotic gene editing and modification. RNAi or RNA interference is a sequence-specific method to silence genes by introducing small double-stranded RNA which mediates with nucleic acids and regulate gene expression. This can be taken as the basic difference between CRISPR and RNAi. CRISPR-Cas is more versatile and superior to RNAi as it can be used to induce insertions and deletions, both repress or activate gene expression, and cause both heritable and non-heritable genomic changes. The specificity is also high in CRISPR-Cas system.

In addition, CRISPR-Cas9 system does not interfere with the endogenous machinery of cell as it is edited at the level of DNA within the nucleus; sometimes it is a major problem with siRNAs or shRNAs, which may lead to cell death. These qualitative advantages of CRISPR-Cas system poise it to effectively dominate RNAi in diverse genetic applications in both clinical and research tool applications fields. However, based on the analysis of the currently available facts, it is reasonable to conclude that RNAi (siRNA, shRNA) will also have a certain unique space in both biomedical research and clinical applications, at least in the near future. The proper use of these two technologies can synergistically bring new value to the use of genetic perturbations for gene function discovery and therapeutic development.

Table 1. Comparison between RNA interference and CRISPR.
 Methods Targeting Sequence Efficiency in Gene Suppression   Knock Down  Knock In Change Genetic Code Change Expression Level Clone Isolation Required
RNAi (siRNA, shRNA) siRNA is the targeting sequence of RNAi Low in CRISPR Yes No No Yes No
CRISPR Synthetic RNA (guide RNA) is the targeting sequence of CRISPR High in RNAi No Yes Yes Yes Yes

siRNA (Small Interfering RNA) Related Information

siRNA (Small Interfering RNA) Related References

1. Dana et al. Molecular Mechanisms and Biological Functions of siRNA. INTERNATIONAL JOURNAL of BIOMEDICAL SCIENCE. June 2017;13(2):48-57.
2. Lam et al. siRNA versus miRNA as Therapeutics for Gene Silencing. Molecular Therapy-Nucleic Acids. 15 September 2015; 4, e252.
3. Ed Davis. Knockout by TALEN or CRISPR vs. Knockdown by shRNA or siRNA.
4. D.D. Rao et al. siRNA vs. shRNA: Similarities and differences. Advanced Drug Delivery Reviews. 61 (2009) 746–759.
5. Boettcher and Mc Manus. Choosing the Right Tool for the Job: RNAi, TALEN or CRISPR. Mol Cell. 2015 May 21; 58(4): 575–585.