Gene knockdown

Methods of gene knockdown

RNA-based RNA interference (RNAi)

Traditionally, there have been two major approaches to decreasing gene expression transiently that involve using RNA to base-pair with host RNA. Although successful, these methods have been challenging, as it is difficult to find a good RNA sequence which interferes strongly with the desired target and not with other RNA sequences. The traditional RNA-based methods are as follows.

siRNA: Small interfering RNA

The siRNA knockdown approach uses a small, exogenous piece of RNA that base-pairs with a complementary target mRNA, leading to degradation of the mRNA. IDT offers Dicer-Substrate Short Interfering RNAs (DsiRNAs), 27mer duplex RNAs that demonstrate increased potency in RNA interference compared to traditional 21mer siRNAs.

shRNA: Short hairpin RNA

This approach uses a small piece of RNA that is converted by cells to siRNA, which then functions just like exogenously-introduced siRNA. shRNA is useful because it can be delivered to cells by viral vectors. This system allows scientists to control the expression of the shRNA with inducible promoters and allows simultaneous delivery of other genes [2]. At high levels, shRNA can cause toxicity to cells [3].

DNA-based RNA interference

Antisense oligonucleotides (ASOs) are DNA oligos, typically 15–25 bases long, designed in antisense orientation to the RNA of interest. Hybridization of the ASO to the target RNA mediates RNase H cleavage of the RNA, which can inhibit the function of non-coding RNAs or prevent protein translation of mRNAs. Steric blocking ASOs can also prevent the RNA from interacting with other proteins including protein translation machinery.

CRISPR-mediated gene knockdown

CRISPR is widely known as a tool useful for complete gene knockout. However, there are also three major ways in which CRISPR can be used to cause transient gene knockdown.  These approaches are as follows:

CRISPRi: CRISPR interference, using dCas9, without additional proteins

dCas9 is directed by guide RNA to a target site in the genome, where it binds but does not cut the DNA. Thus, dCas9 can block transcription when it binds, for example, to the promoter or regulatory region of the targeted gene [4]. The DNA sequence is not altered, so when the cell naturally clears the dCas9 enzyme, the targeted gene is free to be expressed again.

CRISPRi: CRISPR interference, using dCas9, in combination with other proteins

This works by coupling an effector domain (such as Kruppel associated box, KRAB, which binds and represses transcription factors) to dCas9 to repress transcription [5]. The dCas9, while in combination with the effector protein, still is directed by guide RNA to a target gene. The effector protein helps cause a repression of transcription. As with the above method, the DNA sequence is not altered, so when the dCas9 is cleared, the targeted gene is expressed again.

Cas13 family enzymes

Unlike the well-known Cas9 and Cas12a (Cpf1), which target and cut DNA, the Cas13 family of enzymes targets and cuts host RNA and has successfully been used to knock RNA down. However, Cas13 enzymes resemble Cas9 and Cas12a in an important way: they are all directed to their targets by guide RNA. The Cas13 enzymes used for RNA knockdown include Cas13a, Cas13b, and Cas13d [6-8]. Since Cas13 enzymes target RNA rather than DNA, the DNA sequences are not altered. Cellular removal of the Cas13 nucleases will stop their effects on host RNA, allowing normal gene expression again.