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Back to Basics – CRISPR

Recent news has been filled with stories of CRISPR gene editing reaching the clinic, but the technique has been at the forefront of life sciences research for years. In this Back to Basics feature, we provide an overview of the technique for people looking to dip their toes into gene editing.

What is CRISPR?

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, are genetic elements that function as an adaptive immune system in bacteria and archaea. When a host survives an encounter with an invading DNA molecule such as a bacteriophage (virus), it captures a small piece of the viral DNA and incorporates it into its own genome, within what is known as the CRISPR array. The CRISPR array consists of short, palindromic repeats of DNA interspersed with unique spacer sequences derived from the captured viral DNA. The full CRISPR array is transcribed into a long precursor RNA molecule, awaiting processing by host endonucleases.

Processing cleaves the precursor RNA into smaller molecules known as CRISPR RNAs (crRNAs), each containing a spacer sequence. When the organism is exposed to DNA matching one of these spacer sequences, the crRNA guides the CRISPR-associated proteins (Cas proteins) to the exogenous DNA molecule. Cas proteins act as molecular scissors, cleaving the viral DNA at the precise location guided by the crRNA. This interference stage effectively neutralizes the threat by destroying the viral DNA.

CRISPR was first identified in 1987 in E. coli. Over the years, in tandem with increasingly powerful sequencing technology in the wake of the Human Genome Project, the true nature of this bacterial defence mechanism was further elucidated. From the mid-2000s onwards, new advancements came in quick succession, and by 2012, the pieces of this biological puzzle had been put together. It was confirmed that the CRISPR-Cas approach could be exploited and used to alter genomes in a number of organisms, notably in eukaryotic cells. In 2020, CRISPR pioneers Jennifer Doudna and Emmanuel Charpentier won the Nobel Prize for their role in developing the tech.

How is CRISPR used in eukaryotes?

So, how exactly has this system been utilised to provide the groundbreaking therapeutic gene editing powers that you’ve probably heard about?

The key discovery in the CRISPR journey was the revelation that, by creating synthetic guide RNAs complementing a target within a host cell, Cas proteins could be directed to a specific locus, much like when the system recognises a viral sequence. For this purpose, the CRISPR system originally from Streptococcus pyogenes has been applied in a wide range of different organisms, chosen for its simple single protein effector, Cas9.

However, for the system to work there must also be a ‘protospacer adjacent motif’ (PAM), necessary to prevent autoimmunity against the CRISPR locus itself. This limits the target sequences to those that have a PAM directly adjacent to them (a three-nucleotide sequence located at the 3’ end in Cas9 based systems). Despite this requirement in spacer design, the ease with which guide RNAs can be created makes this system much easier to use than alternatives such as zinc finger proteins or TALENS.

After recognition, the Cas protein creates a double-strand break at the target site. In eukaryotes, the cell’s blunt DNA damage repair mechanism, non-homologous end joining (NHEJ), repairs the damage in a highly error prone process. This can then be harnessed to induce gene knockout or introduce specific modifications. This aspect of CRISPR gene editing makes it a particularly useful tool, as the repair of double-strand breaks is key in ensuring the survival of the modified cell, and it allows for further manipulation of the DNA. Additionally, homology-directed repair can be used to knock-in genes, providing a further exciting use.

What relevance does this have?

This technology has widespread applications in genetic engineering, allowing scientists to precisely edit the genomes of various eukaryotic organisms for research, therapeutic and agricultural purposes. CRISPR has become a cornerstone of the lab – allowing researchers to easily knock-out genes to elucidate their roles – and is being widely explored for use in the clinic. A number of clinical trials have explored the tech as a therapy and most recently, the first CRISPR-based gene therapy was approved in the UK and US for sickle cell disease. The drug is known as Casgevy, which precisely alters the genome of patient stem cells to restore normal haemoglobin function. To do this, the stem cells are retrieved from the patient’s bone marrow, altered in a lab using CRISPR and reinfused into the patient’s body. Whilst this may seem like a harsh therapy, the results could be long-term, reducing the need for hospital trips and blood transfusions.

References and further reading:

Addgene. CRISPR Guide. 2023. Available at: https://www.addgene.org/guides/crispr/

Broad Institute. Questions and Answers about CRISPR. 2023. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr

National Human Genome Research Institute. CRISPR. 2024. Available at: https://www.genome.gov/genetics-glossary/CRISPR


More on these topics

CRISPR / Gene Editing / Sickle Cell