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Back to Basics – Base & Prime Editing

In our most recent Back to Basics feature, we took a look at CRISPR, the gene editing technique that took the world by storm in the early 2010s and continues to dominate the life sciences news cycle. But as the years have gone on new methods have come to fruition, which some believe have even more potential.

Two of these techniques are prime editing and base editing. Both pioneered by the lab of David Liu, they offer an exciting alternative to CRISPR and address some of the shortcomings of this method. In this Back to Basics, we take a look at how base and prime editing work and explore some relevant applications.

Base editing

The first paper describing base editing was published in 2016. It enables precise changes to individual nucleotides within the genome without creating double-strand breaks. Unlike CRISPR-Cas9 technologies, which rely on inducing breaks in the DNA strands that are then repaired by the cell’s own repair machinery, base editing directly converts one DNA base to another at a specific target site.

The key components of base editing include a catalytically inactive form of the Cas9 enzyme (known as dCas9) fused to a base-modifying enzyme, such as cytidine deaminase or adenine deaminase. Instead of cutting the DNA, the dCas9-guide RNA complex is guided to the target site in the genome, where the base-modifying enzyme chemically alters a specific nucleotide base, converting it to a different base without disrupting the DNA backbone.

Base editing offers several advantages over traditional CRISPR gene editing. First, it allows for precise single-base changes, such as converting a C-G base pair to a T-A base pair using a cytosine base editor (CBE) or an A-T base pair to a G-C base pair using an adenine base editor (ABE), without introducing insertions or deletions. This precision reduces the risk of unintended mutations or off-target effects associated with CRISPR-Cas9.

Additionally, base editing can efficiently correct point mutations associated with genetic diseases or introduce specific beneficial genetic changes without the need for DNA double-strand breaks and subsequent DNA repair processes, making it a valuable tool for both basic research and potential therapeutic applications. However, base editing’s biggest limitation is that very few types of point mutation can be changed using CBEs or ABEs.

Prime editing

Next out of the Liu lab, in 2019, was prime editing, also referred to as ‘search-and-replace’ editing. Prime editing is another innovative gene editing technique that allows for precise modifications to the DNA sequence, including insertions, deletions and substitutions, without the need for double-strand breaks. It combines the functions of a catalytically impaired Cas9 enzyme (nickase) and a reverse transcriptase enzyme, along with a unique RNA molecule called prime editing guide RNA (pegRNA).

In prime editing, the Cas9 nickase protein is guided to the target DNA sequence by the pegRNA. Once at the target site, the Cas9 nickase creates a single-strand nick in the DNA. The pegRNA contains a template sequence that specifies the desired edit, as well as a primer-binding site for the reverse transcriptase enzyme.

After the nick is made, the reverse transcriptase enzyme uses the template sequence in the pegRNA to synthesise a new DNA strand complementary to the target site. This synthesised DNA strand contains the desired edit. Finally, the cell’s own DNA repair machinery completes the editing process by incorporating the edited DNA strand, resulting in the desired modification to the genome.

Much like with base editing, the lack of double-strand breaks decreases the risk of off-target effects, making it an exciting alternative to CRISPR. Moreover, it improves upon base editing due to the fact that it can make more types of substitutions.

Real-life applications

An exciting real-life application of this tech was highlighted last year. In April, results were published that showed that prime editing was able to efficiently correct the mutation that leads to sickle cell disease – a debilitating blood disorder that primarily impacts individuals of African descent. The disease is caused by a single adenine to thymine mutation in the haemoglobin-Beta gene.

The study followed on from previous work that proved base editing could be used to replace the disease-causing mutation with a harmless variant, yet due to the limitations of the tech, it could not restore the wild-type sequence and function. Using prime editing, however, the team were able to fully correct the mutation in 40% of the patient-derived stem cells used in the experiment. This was a promising result, and subsequent results in mice showed that there was the potential for long-term clinical efficacy.

What next?

No gene editing technique is perfect yet. However, base and prime editing are exciting parts of the genomics toolbox and show great promise for clinical implementation. That said, there is much more to be done to refine the methods before they can be used in humans – scientists from the Broad Institute are working hard to make this a reality. David Liu, the pioneer of both base and prime editing, believes 2024 will be the year we begin to see prime editing make it into human clinical trials.

Whilst it may seem like the benefits of these methods make them much better than tools like CRISPR, it’s important to remember that, despite its ubiquitous nature, CRISPR is still in its infancy too. Ultimately, all of these methods have their unique place in the gene editing field and are only ever being improved.

References and further reading

Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

Scholefield, J., Harrison, P.T. Prime editing – an update on the field. Gene Ther 28, 396–401 (2021).

Lu C, Kuang J, Shao T, et al. Prime Editing: An All-Rounder for Genome Editing. Int J Mol Sci. 2022;23(17):9862 (2022). doi:10.3390/ijms23179862

Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells [published correction appears in Nat Rev Genet. 2018 Oct 19;:]. Nat Rev Genet. 19(12):770-788. (2018). doi:10.1038/s41576-018-0059-1

Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

Kantor A, McClements ME, MacLaren RE. CRISPR-Cas9 DNA Base-Editing and Prime-Editing. Int J Mol Sci. 21(17):6240 (2020). doi:10.3390/ijms21176240

More on these topics

Base Editing / Prime Editing / Sickle Cell