Large DNA Integrations With Prime Editing and Recombinases

What you may not know is that recombinases are originally found in bacteriophages, a type of virus that infects bacteria. These proteins are essential for the bacteriophages’ own propagation within their host. In fact, once they have infected a bacteria, bacteriophages can integrate their own DNA and exploit the replicative machinery of the host cell to make many more copies of themselves.

But how can they do that? Well, thanks to a special class of recombinases known as – drum roll – integrases. Yep, we scientists were low on imagination on that day.

Anyway, these integrases recognize a specific sequence that is present both on the bacteriophage and the bacterial DNA, and catalyze the integration of the bacteriophage’s DNA into the bacterial DNA. Cut and paste again, but with two different pieces instead of one (ages 5-7).

The beauty? This process is very efficient and theoretically it has not limits to the size of what you can integrate

The problem? Human cells do not have a compatible sequence for a bacteriophage Integrase. No “cut here” line, at all. Yeah, we know, that’s good for our sake (no bacteriophages can infect us), but as a genetic engineer it’s kind of a bummer.

However, this was B.C. And those dark times are gone for good!

Team work makes the dream work

Now imagine that you have a new fancy tool – prime editing – which allows you to incorporate edits within the DNA without a double-strand break but has limited integration size.

On the other hand, you have a perfectly evolved machinery to integrate large DNA sequences, the integrases.

It is in the actual name, for CRISPR’s sake!

But, as we said, humans do not have a motif which is compatible with them.

But wait... the necessary sequence is only 46 bp long...which is a piece of cake for prime editing!

If you are facepalming yelling “it was right there!”, that’s a shared feeling.

In fact, two separate groups (Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases - PMC (nih.gov)/Science | Prime Medicine) came up with the same idea: using Prime Editing to incorporate at the desired target site a recognition site for the Bxb1 integrase, called AttB (B stands for “Bacteria”). Yes, indeed, they used prime editing to draw a “cut here” line for the Integrase! Isn’t that genious? Of course, they then delivered the Bxb1 integrase and a DNA template containing the AttP (P stands for “Phage”) and let the integrase do its thing.

And as you can imagine, this worked like a charm. They could integrate, with high efficiency, sequences way larger than before. Hell, they even integrated an entire adenovirus in the genome. That’s a 35 kb – yeah, KB – monster!

But was this all for show? Not really. In fact this technology, known also as Passige (or PASTE, depending on whom of the two teams came first), is currently in Prime Medicine’s pipeline for the development of CAR-T cells using Prime Editing. In this case, Passige will be used to integrate a CAR-T transgene. This will be a great alternative to using standard integrative Lentivirus vectors or a combination of CRISPR/Cas9 and a DNA donor (if you want to learn more about how CAR-T’s are generated and how CRISPR technologies can help that process, check out this episode),

So there you have it, prime editing can now really do it all!

The flaw in the plan:

We know what you’re thinking: There is still a limitation, isn’t it? Otherwise, why shouldn’t we just kick CRISPR/Cas9 to the curb and use Passige instead?

Well, the main problem is that integration through Passige is not completely clean. In fact, during the integration process, we can still retrieve some portions of the AttB/AttP site. Like when you didn’t cut completely over the line and your craft didn’t look exactly right.

Yes, we know you cried. We did too, big time. It’s okay, really.

As you can imagine, if we want to repair a part of a large exon – for example – we can’t do that, because we will also bring in some of the AttB/AttP site. Luckily, there could be a way around it, but if you’re interested to know more, we are happy to discuss it separately, just drop us a message!

All right, that’s all for now regarding prime editing.

As usual, we leave you with a teaser for what’s coming next. Well, we have been away for a bit, so let’s make it two:

1. Prime editing may be not the only one out there, so stay tuned for technologies which can do a similar thing, even though a bit differently.
2. So far both base editing and prime editing were sold as “DSB-free”. But, if you read carefully our articles and the scientific literature, you notice that they do actually break the DNA. And sometimes even a nick can be a problem. Next, time we will talk about this issue and about a true DSB-approach, no excuses!

We hope you enjoyed this episode of Making the Cut and we will see you all very soon!