Prime editing, a step beyond CRISPR

The most precise CRISPR to date turns the genetic scissors into a Swiss army knife 

The CRISPR/Cas9 system, which has already been explained previously in this blog, needs two components, in addition to, of course, the DNA that is going to be modified:

• A protein (Cas9), a nuclease that produces double-stranded DNA breaks.
• An RNA molecule or guide RNA (gRNA): this RNA molecule binds Cas9 at one end and DNA at the other. This DNA binding is very specific and is done to a certain DNA sequence (the sequence of interest) to which Cas9 must bind and cut the adjacent DNA.

Cut and paste

In the classic CRISPR editing, the Cas9 nuclease uses a small RNA molecule as a guide to locate itself in a specific position in the genome, on a specific gene. It then breaks the two DNA strands there after doing one last check.

This awakens the repair systems that are responsible for restoring the continuity of the chromosome. Along the way, we obtain the edition or inactivation of the desired gene, depending on whether or not we provide it with a template DNA that the repair proteins can use to restore the sequence. This is traditional gene editing.

Scissor becomes shuttle

Liu’s team has managed to give this genetic editing system a twist by combining several enzymes as if it were a Swiss army knife [1]. 

They generate a mutant Cas9 by disabling the cutting capacity of Cas9 in one of the two DNA chains. This modified Cas9 is called Cas9n (n stands for nickase, from nick) and, using a scissors analogy, has one of the scissor's edges dull. This enzyme is able to locate itself in the DNA region indicated by the gRNA, but it can only cut one strand [1].

They then disable the cutting ability of the other DNA strand turning the nickase into a dead Cas9, unable to cut the DNA. But it will still be located in the place of the genome that the RNA guide indicates: that opens a world of opportunities. We have turned a pair of scissors into a kind of shuttle or multipurpose module capable of carrying the activity we want to that exact position in the genome. It will be enough to link this new behavior to the inactive Cas9. The simile of the multipurpose knife acquires its full meaning [1].

Initially, Liu’s team fused a fragment of a deaminase enzyme, which can change one DNA letter into another, to an inactive Cas9. More specifically, Liu and his colleagues employed an enzyme that can change C to T. This first CRISPR variant was developed in 2016 and was known as “base editors”, capable of changing certain bases in the genome precisely. With these base editors it was thought that we could treat many congenital diseases by correcting the wrong letters and replacing them with the correct ones, as if it were a molecular corrector, like the famous typex. However, their potential was seriously compromised when it was discovered that they caused numerous changes in unwanted genes [1].

Two for the price of one

After this first attempt, Liu and his team decided to fuse an enzyme called reverse transcriptase (RT) to the Cas9n. In other words, we have a protein capable of copying DNA from RNA at a given site in the genome. Yes, just contrary to the central dogma of molecular biology [2].

Many viruses, including those in the retrovirus family that cause AIDS, use this enzyme to create their genetic material after infecting cells. These retroviruses carry RNA as their genetic material, and the first thing they do after infecting a cell is to synthesize DNA from that RNA. Once the viral DNA has been synthesized, they start using it regularly.

What could it be used for? to direct the DNA copy that we want to produce according to the information contained in the RNA that acts as a template [2].

How did they get the RNA to act as a template? Really simple. They came up with the concept of lengthening the short guide RNA molecule, which is responsible for positioning the Cas9n at a location in the genome. The other strand of DNA can now use that new end as a template [2].

Just to point out that, despite the fact that fusing enzymes may sound like something out of science fiction, it is actually a relatively frequent process in genetic engineering. An enzyme is nothing more than a chain of amino acids.

The idea of ​​fusing these two enzymes is revolutionary and intelligent, and allow to make a virtue out of necessity, since we now use the same RNA molecule for two things:

• One end serves to pair with one of the two DNA strands and thus position Cas9 in the desired place in the genome.
• The other end serves as a template to direct the synthesis of the other DNA strand, the one we have cut. We can direct the synthesis from the sequence that we put on that new end of the RNA.

Thus, a mutation can be fixed by adding the right letters. Or, vice versa, generate it if you want to know what happens when that gene is altered.

Prime editing

They have called this new capability of the CRISPR tools "prime editing" (PE), which plays with the dual meaning of "quality editing" and "guided by a mold". The authors claim that prime editing can theoretically correct up to 89% of the more than 75,000 genetic errors that lead to disease in human beings. 

You may wonder, "Why that number?" Well, because this tool is designed to modify small or point mutations that constitute 89% of those that are known. Remember that the length of the guide-template RNA itself determines the editing capacity, which cannot be "kilometric."

So far we know, thanks to Liu's work, that PE functions in cultured human cells, though not in all cell types equally. It is crucial to remember that:

1. The designed changes are efficiently achieved
2. The unpredictability of the outcomes and the occurrence of unintended mutations in other regions of the genome are significantly reduced.

At ZeClinics we are committed to improve the potential of prime editing tools. We have recently been granted €1M from the EIC-Pathfinder Challenge for the EdiGenT project to develop new prime editing and non-viral delivery strategies in zebrafish. Our goal is to address the four major challenges that gene therapy approaches currently face.

REFERENCES

[1] Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May 19;533(7603):420-4. doi: 10.1038/nature17946.

[2] Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785):149-157. doi: 10.1038/s41586-019-1711-4.

By Miriam Martínez

Miriam is a Human Biologist expert in neuropharmacology. After a master’s degree in Pharmaceutical and Biotech Industry, she obtained her PhD in Biomedicine from Pompeu Fabra University (Barcelona). During her doctorate, she focused her research on the behavioral analysis of animal models for neurophenotypical characterization. Since then, she has been working in the healthcare marketing and publicity sector, where she has contributed to developing marketing campaigns for several pharmaceutical brands. In 2021, she joined ZeClinics with a branding and marketing strategy focus.