Prime Editing: The Future of Genetic Engineering

In 2012, when it was discovered that a protein called Cas9 (CRISPR associated protein 9) could be used to edit genes with never-before-seen precision, there was instantly widespread discussion of the new possibility of curing innumerable genetic diseases, but also of designer babies, and genetically engineered super-people. However, CRISPR-Cas9 gene-editing doesn’t have the precision necessary to make the kinds of changes to cure many diseases — at least not without some collateral damage. But in 2019, an article in Nature outlined a new “search-and-replace” gene-editing technology that offered solutions to some of CRISPR’s biggest flaws. Although very new, this technique called “prime editing” looks particularly promising and could revolutionize our understanding of the potential of gene-editing in the very near future.

What is CRISPR?

The idea of gene-editing didn’t first appear in 2012; ever since the discovery of DNA’s structure in the 1950s, people have been trying to figure out how to manipulate DNA to cure or prevent disease, or to alter certain traits. Prior to the discovery of CRISPR, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were used for gene-editing. These are both fusion proteins that bind to a target stretch of DNA and cut both strands of it. However, unlike ZFNs and TALENs, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, uses guide RNAs (gRNAs), which are exactly what they sound like — single-stranded sequences of genetic information that guide Cas9, a protein that defends bacteria against DNA-viruses, to a target DNA sequence, where the Cas9 essentially acts like a pair of scissors, cutting the two strands.

After this cut is made, the cell repairs the DNA through one of two methods: Non-Homologous End Joining (NHEJ), and Homology Directed Repair (HDR), with the former being more common. In NHEJ, the DNA on either side of the Cas9-induced break is simply stitched back together. The most common form of HDR is called Homologous Recombination (HR). HR patches the break by copying the template of a homologous gene; one that serves the same function, but ideally does not have the same undesirable mutation as the DNA did previously. NHEJ is more common than HR because it is more efficient and does not require as much information. However, HR is a much more precise method of repair, given that NHEJ is prone to creating new mutations, and HR is therefore more desirable for CRISPR editing. The fact that cells do not always repair the double-strand breaks created by Cas9 in this way makes CRISPR an unreliable editing system that frequently damages the target gene and the DNA around it.

What about Prime Editing?

Prime editing, in a lot of ways, is very similar to CRISPR. It follows the same formula of “find the mutation, slice the DNA.” It also requires the same basic components: a DNA-cutting enzyme, or endonuclease (like Cas9, for example), and a single-stranded guide RNA. However, it has a few major changes that make it much more precise, and less likely to introduce dangerous mutations.

Basically, prime editing is CRISPR without its biggest shortcoming: the double-stranded breaks. Prime editing avoids the mess created by simply hacking through two strands of DNA by using a variant of Cas9 called Cas9 nickase. As the name suggests, Cas9 nickase acts less like scissors, and more like an X-Acto knife, and nicks the target DNA, rather than creating a complete break. Attached to the Cas9 nickase is another enzyme, called reverse transcriptase, and a modified version of CRISPR’s gRNA, creatively titled prime editing guide RNA, or pegRNA. Altogether, these three components make up a prime editor pegRNA complex, or PE:pegRNA complex for short.

Much like Cas9 did in CRISPR editing, the PE:pegRNA complex joins to the target stretch of DNA. There, the Cas9 nickase nicks only one strand of the mutated DNA. Then the reverse transcriptase does its job and reverse transcribes the edited RNA found in the pegRNA, replacing the target stretch of DNA, which is detached from the chromosome. An additional gRNA fixes the unedited strand of DNA to match its counterpart, the nick is repaired to match the newly edited DNA, and bam: DNA repair without the mess of a double-strand break.

Why is Prime Editing Important?

CRISPR’s lack of precision and its propensity to create unwanted mutations are a big deal, and mean that CRISPR’s liable to cause genetic diseases, or even create mutations that trigger cancer. There are other methods of gene-editing that are less risky, such as base editing, which replaces individual nucleotides, but base editing has a relatively limited scope, and can only correct four of the twelve types of point mutations, whereas prime editing can correct all twelve. This scope and precision means that prime editing is theoretically capable of correcting about 89% of all genetic mutations known to cause disease in humans, a staggering number that would revolutionize the treatment of genetic diseases and make many obsolete.

Prime editing’s capabilities aren’t just hypothetical, either — it has already been used to correct two genetic diseases in lab-grown human cells: sickle cell anemia, which is caused by a single nucleotide mutation, and Tay-Sachs Disease, which required the correction of a longer stretch of nucleotides. Both of these diseases are currently considered to be incurable, and children with Tay-Sachs disease rarely live past the age of four. This means a cure for either of these diseases would be huge, let alone a precise, highly efficient method of gene-editing that can cure these diseases and many more.

What Does the Future of Prime Editing Look Like?

Prime editing is in its infancy, and there’s a lot more research to be done before it can be used to treat humans. Although it is certainly more efficient than CRISPR in most cell types, it still has many limitations. Researchers need to do animal tests to find a method by which PE:pegRNA complexes could be safely, effectively delivered to human cells. One idea is that it could be encapsulated in a virus and enter a cell that way, but that’s only one of multiple methods that are being considered. It is also unknown whether or not prime editing will trigger an immune response. Reverse transcriptase is actually an enzyme used by RNA viruses, and cas9 nickase and pegRNA are both variants of macromolecules that originated in bacterial cells, which means the immune system would have every reason to reject PE:pegRNA complexes. Another issue is that currently, prime editing is only about 20–50% efficient, depending on cell type, which just isn’t going to cut it for diseases caused by multiple highly specific mutations in a single gene, such as cystic fibrosis.

However, once these problems are solved, it’s likely that prime editing will become an important gene therapy tool. Although it is not likely that it will replace CRISPR or base editing, since all three methods have their advantages, it will certainly take its place alongside them as a treatment option for a multitude of genetic disorders and is advantageous enough that it may become the go-to. While the possibilities of prime editing are almost limitless — it has been suggested that it could be used to remove cancer-causing mutations, engineer new foods, or even to fight viruses and bacteria by dismantling their genetic code — what is certain is that more research will be done, and that we might be closer than we realize to a future where people with diseases previously thought to be incurable are given a shot or a bottle of pills, not a hopeless diagnosis.

Key Takeaways

• CRISPR-Cas9 editing is unreliable, and double-strand breaks create damage that can lead to unwanted mutations
• Prime editing is similar to CRISPR, but uses small nicks instead of double-strand breaks to minimize damage
• Prime editing has already been used to treat sickle cell anemia and Tay-Sachs Disease in human cells, and can theoretically treat 89% of mutations that cause genetic diseases in humans
• While there’s still a lot of research to be done, prime editing is a precise, efficient gene-editing tool that could be commonly used in gene therapy in the near future