Gene Silencing – RNAi or CRISPR?

Regardless of your research area, there may come a time when you need to uncover the function of an unknown protein or gain deeper insights into the mechanisms of a cellular pathway. This can be achieved through various strategies: targeting the protein level using small molecule inhibitors or blocking antibodies, modifying the gene sequence encoding the protein of interest, or interfering with RNA transcripts at the transcriptional level.

This article explores CRISPR and RNA interference (RNAi), two widely used methods for investigating gene function, and compares their advantages and limitations in a laboratory research context.

First, let’s recap on CRISPR and RNAi

CRISPR

With roots in an ancient prokaryotic immune system, CRISPR is one of the hottest gene-editing tools around today.

In brief, CRISPR involves the use of a Cas endonuclease – often Cas9 – and a guide RNA (gRNA). The gRNA is specially designed to have sequence overlap with the target region. It guides the Cas to the desired target site, where the Cas makes a double-stranded break (DSB) that can be repaired through either the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway.

With conventional CRISPR, it is possible to knock in and knock out genes/sequences of interest or mutate target sequences, and the resulting changes to the genome are permanent.

RNAi

While CRISPR makes permanent genetic changes by altering the genomic DNA sequence, RNAi silences genes temporarily by knocking down their mRNA transcripts in an antisense manner.

RNAi is also a naturally occurring process. It was first discovered in the nematode Caenorhabditis elegans, when researchers at the University of Massachusetts Medical School encountered a small non-coding RNA (which is now known as a microRNA) that could modulate aspects of post-embryonic developments by binding to a complementary repeated sequence element in the 3′ untranslated region (UTR) of a gene involved in deciding stage-specific cell fates in Caenorhabditis elegans.

Nowadays, RNAi is widely used in research lab and within the development of new therapies, where exogenous small interfering RNAs (siRNAs) are used to silence mRNA transcripts using already existing cellular machinery.

Whether naturally-occurring or provoked by the addition of exogenous siRNAs, RNAi relies on processing machinery that is already present within all cells.

CRISPR vs. RNAi – Which tool is best?

CRISPR and RNAi are both widely used in research and clinical development for new therapies, and their overall importance for science has been recognised with the awarding of Nobel Prizes to the scientists behind both techniques. But when it comes down to it, how do they compare in the lab with respect to workflow, advantages and drawbacks? For convenience, we’ve compiled lists of the main pros and cons of each approach below.

CRISPR

Pros

Permanent knockout: because CRISPR makes changes to the DNA, the effects are irreversible. This may be desirable for the production of sets of stable cell lines bearing different mutations in your gene(s) of interest.
Complete knockout: because it is possible to completely eliminate a gene (by deletion) or render a gene completely non-functional (by engineering a premature stop codon or deleterious mutation) with CRISPR, there is no risk that residual target gene expression can confound experimental results.
Specific: in its early days, CRISPR-Cas suffered some non-specificity caused by off-target cleavage. Today, massive improvements to gRNA design tools, the development of chemically modified RNAs and the emergence of new Cas variants with increased specificity has greatly improved the precision of gene editing with CRISPR-Cas.
Rapidly evolving toolbox: CRISPR is a rapidly evolving tool with new variations emerging continuously. Although not discussed here, RNA-targeting Cas endonucleases, e.g., Cas13, have been discovered, and these facilitate similar gene silencing outcomes to RNAi, with demonstrated effect in mammalian cells (1).

Cons

Not possible to study essential genes: Since deletion of an essential gene is lethal to the cell/organism, conventional CRISPR is not a suitable choice for the study of essential genes. Note however that this may be possible with a modified CRISPR workflow known as CRISPR inhibition (CRISPRi), using a catalytically dead Cas9 and a gRNA to modulate transcriptional processes at the DNA level (2).

RNAi

Pros

Possible to study essential gene function: dose-responsive gene silencing is possible with RNAi, making it possible to study the function of essential genes by titrating mRNA transcript levels and studying resulting phenotypes. Dose-responsive gene silencing may also be attractive for the study of non-essential genes, e.g., to study the effects of different extents of silencing of a gene of interest and to hone in on whether other genes can compensate when your gene of interest is silenced.
Transient knockdown studies possible: again, because RNAi does not make permanent genetic changes, it allows the study of transient gene silencing which may be useful in validating experimental observations through restoration of phenotype when the gene silencing effects wear off.
Fast and easy workflow using easily accessible reagents: transfection with siRNA to study gene function is often quicker and simpler than a CRISPR workflow, which usually requires cloning of the Cas and/or gRNA sequences into plasmid vectors.
Better representation of therapeutic effect: in some cases, gene knockdown as opposed to knockout might better recapitulate the phenotype(s) caused by an experimental drug or inhibitor, where the latter might not completely eliminate target protein function but reduce it to a level that is pharmacologically beneficial.

Cons

Off-target effects: one of the biggest downsides of RNAi is that it suffers from sequence-independent and sequence-dependent off-target effects. This poses a risk of skewed results particularly in screening experiments that involve silencing of large numbers of genes in parallel, where it may not be feasible to control for off-target effects for every siRNA used. Although CRISPR is not 100 % precise, a recent analysis found that CRISPR is less prone to off-target effects than RNAi (3).
Incomplete knockdown may lead to ambiguous results: not all siRNAs are equally efficient at knocking down their targets. In some cases, poor knockdown may confound experimental results leading to ambiguity, resulting in the (possibly time-consuming) search for alternative siRNAs.

The verdict

As with most aspects of biology, there’s no definitive answer when comparing CRISPR and RNAi. This post provides an overview of the key advantages and limitations of each approach to gene interrogation, aiming to help you decide which method best suits your needs.

Ultimately, the choice depends on your experimental objectives. For some studies, partial gene knockdown may be more appropriate to address the research question, while in other cases, a complete knockout might be necessary if even trace amounts of functional mRNA can sustain biological activity.

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