No matter what area of research you are working in, at some point or another you may need to decipher the function of a completely unknown protein or better understand the mechanisms of a cellular pathway.
There are many ways of going about this, either by interfering at the protein level through the use of small molecule inhibitors or blocking antibodies, the genetic level by altering the gene sequence that encodes your protein of interest, or at the transcriptional level by interfering with the RNA transcript.
This piece looks at CRISPR and RNA interference (RNAi) – two commonly used methods to interrogate gene function – and compares the pros and cons of each approach within a lab research setting.
First, Let’s Recap on CRISPR and RNAi
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. You can read about the history of CRISPR and its major applications today in a previous blogpost.
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. You can read more about how RNAi works in our previous blog post.
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 and Cons
- 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).
- 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 and Cons
- 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.
- 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.
It’s Not Black and White
Like almost everything else in biology, there is no black and white answer when it comes to CRISPR vs. RNAi. In this post, we have tried to give an overview of the most important pros and cons for each approach to gene interrogation. These will hopefully help you to consider which approach is right for you.
In reality, the choice will depend largely on the experimental goal – maybe partial gene knockdown allows one to address their research question better than knockout, or maybe complete knockout is needed because even trace amounts of functional mRNA are sufficient for biological function.
Get in Touch!
Do you have other pros and cons that you would like to share? We would love to hear them so that we can share them with all of our readers. You can write to us anytime at firstname.lastname@example.org.
Related Nordic BioSite Blog Posts:
Step aside CRISPR, RNA editing is taking off: News Feature in Nature that provides an overview of the status of RNA-based CRISPR editing (2020).
- D. B. T. Cox et al., RNA editing with CRISPR-Cas13. Science 358, 1019-1027 (2017).
- L. S. Qi et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183 (2013).
- I. Smith et al., Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map. PLoS Biol 15, e2003213 (2017).