CRISPR-Cas9: The genius genome editing system

3rd May 2022 - Last modified 19th October 2023

20 years of Alto. 20 years of science. #1

By Peter Cussell, PhD, Science writer

As part of Alto Marketing’s 20 year celebrations, we’re looking back at some of the most important advances in science over this time in our blog series “20 years of Alto. 20 years of science.” And what better way to start than talking about the genius genome editing system that is CRISPR-Cas9!

Over the past two decades genome editing has materialised from science fiction to reality and is today provoking profound changes in biomedical research and medicine alike. More than any other discovery in this field, the refinement of the CRISPR-Cas9 system represents the greatest technological leap forward in genome editing.

Recent years have seen an exponential rise in CRISPR-Cas9 research, and this revolutionary development was acknowledged in 2020, when the Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna; co-authors of the breakthrough paper in Science detailing the CRISPR-Cas9 system [1].

Now the technology has been refined, CRISPR-Cas9 is being adopted by clinical and research settings across the globe and is evoking an ever-growing and versatile list of applications. But what exactly is CRISPR-Cas9 and how (and why) has it become so popular?

A bacterial barricade…

When investigating the function of a gene, one of the most direct methods would be to block its expression completely and then analyse the outcomes. There are several methods towards “silencing” a gene of interest, and before the adaptation of the CRISPR-Cas9 system, RNA interference (RNAi) was the go-to choice for researchers.

RNAi is a post-transcriptional intervention that relies upon specific double-stranded RNA molecules that target and bind in a complementary fashion to specific messenger RNA (mRNA) transcribed from the gene of interest to block its translation into protein [2]. While RNAi is an inexpensive and easy-to-adopt method, there are some significant drawbacks with this technique.

In an in vitro model, it is difficult to transfect every cell in a population using RNAi and so some level of gene expression still occurs. Furthermore, RNAi only inhibits gene functionality transiently; meaning that researchers only have a short window of opportunity to perform assays before protein expression returns to natural levels. Such issues are inevitable when attempting to disrupt gene expression at the RNA level and often produce results limited in replicability.

A more optimal approach towards gene silencing would be to target the gene of interest at the DNA level, and this breakthrough was made when Charpentier and Doudna identified the CRISPR-Cas system [1]. Short for ‘clustered regularly interspaced short palindromic repeats’, the CRISPR-Cas system was initially identified in bacteria. Charpentier and Doudna described CRISPR as a prokaryotic immune mechanism that provides bacteria with memory of viral infection and defence against reinfection. The CRISPR-Cas system integrates the viral DNA into the bacterial genome, where the Cas nuclease can remain primed to target and cleave the viral sequences upon future infection. Researchers have adapted the CRISPR system into a powerful gene editing tool that can make targeted gene deletions and replacements to the DNA of any organism. Complexing a Cas9 nuclease with a specific guide RNA (gRNA) directs the Cas9 to the target sequence to perform a cleavage at the target site. This has enabled researchers to induce full gene knockout and provides the ability to silence multiple genes simultaneously with the addition of multiple gRNAs. Thus, CRISPR-Cas9 represents a more efficient gene silencing tool compared with RNAi, while also remaining easy-to-use and low-cost.

How can CRISPR-Cas9 be harnessed for biomedical research purposes?

One of the most widely adopted uses of CRISPR-Cas9 is to regulate endogenous gene expression. While the effects of being able to affect gene expression may seem obvious at first, the array of opportunities this presents is huge, allowing researchers to regulate the expression of proteins such as receptors, enzymes and transport proteins; facilitating the discovery of novel cell signalling components and drug targets.

In addition to CRISPR-Cas9’s gene knockout capabilities, the platform can also be harnessed to insert, or “knock-in” a DNA sequence of choice into a genome. CRISPR-Cas9 DNA insertions include modified genes, exogenous reporters (e.g., fluorescent proteins) and single nucleotide polymorphisms.

CRISPR-Cas9 has facilitated a deeper understanding of individual gene functionality, developmental pathways and disease pathologies. The development of a Cas9 nuclease lacking the catalytic domain for DNA cleavage has allowed researchers to utilise CRISPR-Cas9 to activate (CRISPRa), or inhibit (CRISPRi) a gene of interest without requiring genome modification [3]. Substantial breakthroughs have been made in the field of disease modelling thanks to the adoption of CRISPR-Cas9, where researchers have been able to create accurate cell and animal disease models that span beyond genetic disorders and include cancers, neurological conditions, immunodeficiencies, cardiovascular conditions and infectious diseases.

What about the potential clinical applications of CRISPR?

The prospect of utilising CRISPR-Cas9 for the treatment of human diseases is edging ever-closer, and the demand for new interventions has never been greater. It has been estimated that approximately 10,000 human diseases are caused by a mutation in a single gene [4], and the majority of these have no clinically approved options to speak of. Current clinical applications utilise knock-in and knock-out features of CRISPR, and fall into two broad categories: ex vivo – whereby cells are genetically modified outside a patient’s body and transplanted back into the patient; or in vivo – which involves modifying cells inside the body, requiring complex delivery systems.

Ex vivo approaches have proved highly effective in the treatment of cancers, where CRISPR-Cas9 has been harnessed to improve the efficacy of engineered T-cells to destroy tumour tissue. With targeted knockout of the T-cell’s programmed cell death protein (PD-1) their longevity and effectiveness at fighting cancer is significantly improved. Ex vivo CRISPR interventions in development include those for leukaemia, HPV-related cancers, lung and various other cancer types as well as HIV [5].

In vivo CRISPR applications are proving more difficult, and generally rely upon viral vector or nanoparticle delivery systems to target the tissue of interest. Because of the challenges with delivery, most trials underway target more easily accessible tissues such as the cervix, eye and liver. Various in vivo animal studies have successfully demonstrated the correction of a disease using CRISPR-Cas9. In one study, mice with hereditary type-1 tyrosinemia were cured by correcting a faulty gene linked to a liver enzyme deficiency [6]. In the future, CRISPR is tipped to pave the way towards viable treatments for a host of currently untreatable genetic disorders from sickle cell disease to cystic fibrosis. The prospect of germline editing and CRISPR babies does bring up some profound ethical issues however, with the line between disease intervention and eugenics difficult to draw. A unilateral and scientific approach therefore will be required to ensure that CRISPR is used in a responsible manner.

Summary

We have seen how CRISPR-Cas9 is disrupting the world of biomedical and clinical research alike, and despite CRISPR having revolutionised many aspects of genetic research, the technology is still in its relative infancy. Who knows what the next 10 years will bring?

At Alto, we love science! With six PhDs in our team, writing about science and communicating the benefits of techniques, products or services is what we do best. Combine this scientific knowledge with many years of experience in life science and healthcare marketing and you can be sure that your content and communications are in safe hands. Whether you’re looking for a detailed white paper summarising the latest developments in your field, an interview with researchers using your products in the laboratory or a catchy blog for your website, get in touch as we would love to help!

References

1. Jinek, M. et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337 6096, 816-821 (2012).

2. Han H. RNA Interference to Knock Down Gene Expression. Methods Mol. Biol. 2018, 293-302 (2018).

3. Memi, F. et al. CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations. Seminars in Perinatology. 42 487-500 (2018).

4. Konishi, C. et al. Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases. J. Biomed. Res. 35 2 148 (2021).

5. Hirakawa, M. et al. Gene editing and CRISPR in the clinic: current and future perspectives. Biosci. Rep. 40 4 (2020).

6. Yin, H. et al.(2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. biotechnol. 32 6 551–553 (2014).