At the cutting edge

Since the discovery of CRISPR technology, research into genomic science and novel therapy development has exploded. Rebecca Laborde* explains the science behind CRISPR and how it is encouraging research innovation.

Rapidly developing genomic technologies have become a cornerstone of modern medicine and novel therapy development. In the past two decades, they have moved from obscure research laboratories into the mainstream, making DNA testing accessible to the public.

Media coverage surrounding the genome editing technology called CRISPR (clusters of regularly interspaced short palindromic repeats — pronounced crisper) has led to strong interest from both experts and interested lay-people in its medical application and use in developing innovative technologies.

The natural origins of CRISPR

CRISPR is the popular name for a genome editing technique that has been adapted from a naturally occurring function of single-celled bacteria. Bacteria are frequently invaded by viruses that enter the bacterial cell and cause a variety of negative effects. To protect themselves, the bacteria employ an immune defence system capable of cutting the viral genome into sections and rendering it inactive.

This is possible because of a naturally occurring defence mechanism in bacteria called CRISPR and an enzyme called Cas9. The basic mechanism involves these clusters of repeated patterns within the bacterial DNA being used as a template to produce a small tag of complementary genomic material (RNA). This tag guides the bacterial Cas9 enzyme to the complementary tag on the viral genome, causing the viral DNA to be cut apart and neutralising the negative impact of the virus.

CRISPR as a genome editing technology

While researchers have been aware of CRISPR repeat regions for over 30 years, it was not until a 2007 study by Barrangou et al that their potential became clearer¹. This group was working with Streptococcus thermophilus, a bacterial species commonly associated with dairy products. These experiments involved exposing the bacterial cultures to various types of viruses and then monitoring the formation of new palindromic repeat regions (CRISPR regions) into the bacterial genome.

They identified that subsequent exposure to the same virus resulted in viral genome cleavage and inactivation of the virus. Further, they demonstrated that if they removed the viral specific CRISPR region and replaced it with a different viral specific sequence, they could alter the immunity of the bacteria. This important study opened the possibility of converting this bacterial immune component into a tool for genome science.

A variety of groups began working to adapt this bacterial defence mechanism into a technology that could be applied for directed genome manipulation. A 2012 study by Jinek et al described the ability to produce an RNA molecule in the lab that complements a desired genomic sequence². This custom crRNA molecule was fused to a component called a tracerRNA to produce a “guide RNA”. This guide RNA allowed scientists to specifically designate the region of a genome (such as human, plant or animal DNA) that will be cleaved by the Cas9 enzyme.

Additional steps in the process for introducing DNA sequence back into the genome rely on the normal DNA repair mechanisms of the cell. This allows scientists to supply a small piece of template DNA to be copied into the area that is being repaired, resulting in the inclusion of that sequence into the genome at the location targeted.

Where is CRISPR being applied?

If you perform a web search today for “CRISPR applications” you will find more than three million results spanning wide varieties of medical treatment approaches, research applications on every species of organism imaginable and products including agricultural and genome-edited foods. The relative simplicity of the technology and reasonable cost for application has rendered this one of the most exciting developments in genome science. Productised solutions for applying CRISPR technology are widely available through an array of commercial providers. This is driving a rapid adoption of this technology into most genomic science programs.

The future of CRISPR technology in medicine

The benefits of targeting genome editing technology in medical research are evident both in the ability to create mutations for research purposes and the ability to remove destructive mutations or replace mutations with normal sections of genes for the clinical treatment or cure of disease. A recent review provides an overview of the areas of medical research already targeting the treatment of genetically based human diseases including cystic fibrosis, Fanoni anemia and cataract retinitis pigmentosa (RP), a genetic disorder causing retinal degradation and blindness³.

Other areas in medicine likely to significantly benefit from CRISPR technology include cancer immunology and stem cell research and many others. Additional diseases may be targeted by preventing disease transmission such as vector control via modification of the genome for mosquito populations that harbour malaria. The options for application of CRISPR technology are diverse and we are only at the beginning of what may be possible for the future of genome science.

A bit of caution for CRISPR

We are clearly standing at the edge of a period of great discovery and advancement in medical genome science. It is expected that an increasing percentage of newly initiated clinical trials for genomics diseases in late 2018 and beyond will include a component of CRISPR derived therapy. This rapid pace of discovery and development fuelled by CRISPR merits a healthy level of caution in developing new applications. Despite recent advances to further improve the specificity of CRISPR technology⁴, this technology is not guaranteed to be 100% efficient in all cases.

Variability is a key component of natural genomes and leads to the possibility of CRISPR applications having off-target effects and impacting the patient in unexpected ways. While the technology continues to improve, we must be diligent to use best practices in its application to human medicine. Additional concerns for future generations include ensuring we develop science-based standards on the logical and ethical use of this technology to treat patients and produce products.

There is a great responsibility placed on the medical and scientific community to apply these powerful tools responsibly. The public also plays an important role in having an educated understanding of the risks and potential benefits of these applications so that we may all work together to move medicine forward in this era of genomic medicine.

*Dr Rebecca Laborde is Lead Product Strategist for Healthcare and Master Principal Scientist for Precision Medicine at Oracle Healthcare.

References:

1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007 Mar 23;315(5819):1709-12. PubMed PMID: 17379808.

2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A, programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21. doi:10.1126/science.1225829. Epub 2012 Jun 28. PubMed PMID: 22745249.

3. Fellmann C, Gowen BG, Lin PC, Doudna JA, Corn JE. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nat Rev Drug Discov. 2017 Feb;16(2):89-100. doi: 10.1038/nrd.2016.238. Epub 2016 Dec 2 Review. PubMed PMID: 28008168; PubMed Central PMCID: PMC5459481.

4. Jia Y, Xu RG, Ren X, Ewen-Campen B, Rajakumar R, Zirin J, Yang-Zhou D, Zhu R, Wang F, Mao D, Peng P, Qiao HH, Wang X, Liu LP, Xu B, Ji JY, Liu Q, Sun J, Perrimon N, Ni JQ. Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM. Proc Natl Acad Sci USA. 2018 May 1;115(18):4719-472 doi: 10.1073/pnas.1800677115. Epub 2018 Apr 16. PubMed PMID: 29666231.

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