Ideation, Iteration, Innovation At the Frontier of Genome Editing (pt. 3 of 3)

Prime Editing is the latest breakthrough in genome editing and adds a potential tool to the CRISPR-Cas9 toolkit

From an article on gene editing from The Conversation

Contents

1. Introducing to the World: Prime Editing (pt. 1)
2. DNA and RNA: A Refresher (pt. 1)
3. Editing The Genome: Enter CRISPR-Cas9 (pt. 1)
4. A Complementary Technique to CRISPR-Cas9: Base Editing (pt. 2)
5. An Evolved Version of Base Editing: Prime Editing (pt. 2)
6. Why This Is Groundbreaking: Word Processors (pt. 3)
7. A Final Word On Ideation, Iteration and Innovation (pt. 3)
8. 🔑 Takeaways (pt. 3)

Return to pt. 1 or pt. 2.

6. Why This Is Groundbreaking: Word Processors

Randall Munroe from XKCD somehow always a brilliant and funny way to underscore how things work, and genome editing is no exception

This is how I think about each from the perspective of a word processor like Microsoft Word.

All three techniques have a Find (Ctrl + F) function that allow you to look. None of the three techniques search for text directly; they all look for specific area that is next to the text. However, the pegRNA (prime editing guide) used with Prime Editing allows for search in more contexts and with fewer constraints than search via Base Editing and traditional CRISPR-Cas9 editing.

But the real power lies in this:

1. CRISPR Cas9 grants you the ability to do Search and Replace, Insert or Delete but you can only make changes in certain types of files (e.g., files that can be copied and pasted). You cannot use it with sufficient reliability or control via NHEJ (and not at all with HDR) in existing files that have already proliferated and can no longer be copied — the equivalent of not being able to use it to impact post-mitotic cells in adult humans like neurons.
   The other issue is that you know how sometimes you have the same sequence that you look for in a text file but DON’T want to modify?
   Or, let’s say you want to edit the same thing in multiple cells at once? It will edit in some, and not in others, with lower efficiency than Base Editing (for its specific narrow case) and than Prime Editing (in a number of the cases tested thusfar).
   The advantage though of CRISPR Cas9 is that it’s been tested in real humans, so it’s as if your word processor is a real live product being sold on the market rather than just on a beta or early version.
2. Base Editing allows you to only make two specific types of Search and Replace functions, e.g., switching a C-G pair for an A-T or T-A pair, or an A-T pair for a C-G or G-C pair. But when you want to change all of them or it doesn’t matter which ones you change within a certain length of text, Base Editing is the way to go!
3. Prime Editing allows you the most flexibility and efficiency. It works like a full word processor, complete with the ability to Search and Replace, Insert or Delete (and even combinations of them). It allows you to make larger deletions or insertions with greater efficiency and fewer mistakes or byproducts (e.g., random letters or words inserted where you don’t want). At least one limitation not captured above is that Prime Editor — because of how big it is — might only be limited to files of a certain size.

7. A Final Word On Ideation, Iteration and Innovation

Pict from Telegraph, although this article on research as a maze using only local info is far more instructive.

The background to ideation and iteration that led to this innovation is illustrative. Many endeavors — whether scientific research or building a successful company — is full of dead ends, we don’t often discuss the ideas that don’t seem worth pursuing, or the ones that we do pursue but don’t yield the results we hypothesized. Sometimes these dead ends are worth publishing so others know not to pursue them (and this does happen from time-to-time), but there are far fewer failed experiments and startups we have not heard of. What makes them successful is not analyzed or broken down properly. Nor is luck properly attributed its due in these circumstances.

As David R Liu broke down in this interview when someone asked what do you consider to be the key inventive step in the prime editing system, there were a few key questions and considerations:

• Background knowledge and limitations of existing technologies (e.g., his lab was well-positioned since they had helped research and develop base editing techniques).
• Define the precise problem that you want to address, and ideate potential ways to do so (e.g., can we develop a technique that finds a target site in the genome and directly writes new strings of DNA letters that can replace the original DNA sequence).
• Leveraging knowledge and expertise outside your own domain and knowledge, using the existing body of knowledge to find ideas that are potentially suited to addressing your problem (e.g., combining knowledge from biochemistry on CRISPR-Cas9 and reverse transcriptases and knowledge from bioengineering).
• After ideation, comes iteration through testing and updating understanding, synthesis, brainstorming. They had to evolve and update their approaches as they found things that worked or didn’t work according to their original hypotheses, and determine if these were fundamental barriers or other ways to work around these issues. They are also working to find ways to tweak to improve outcomes and results — in terms of effectiveness, efficiency and unwanted byproducts.
• After a few rounds of testing in test tubes, e.g., in vitro and yeast, they started testing prime editing in mammalian cells; their initial mammalian cell experiments yielded no editing at all, and it wasn’t clear if the problem was one we could overcome or if it instead was an intrinsic feature of mammalian cells; ultimately, they strengthened the primer-template complex, yielding the first low-efficiency but unequivocal prime editing in human cells, thus validating that mammalian cells could indeed support all the steps and there was no fundamental feature that would limit the potential of this technique.
• There were also undoubtedly many moments of luck, serendipity and flip the coin let’s just try this moments he left out.
• Finally, as you can see, there are 11 names included. This paper undoubtedly represents quite a few years of research and many rounds of experiments, but there is a moment when you feel the ground on which your ideas stand is solid enough and it’s worth sharing the results with the world. This is a key step on the path to innovation. Many more tests still need to be done and hoops must be jumped in order for prime editing to have the same depth of knowledge, application, possibility as base editing and classical CRISPR-Cas9 genome editing. The possibility that this will represent a true breakthrough will require the test of time, but the potential is exciting nonetheless as researchers and scientists are undoubtedly working to continue exploring, testing, refining the technique in a wider array of situations.

8. 🔑 Takeaways

• Prime Editing enables search and replace genome editing without double-strand breaks or donor templates.
• Prime Editing works by combining targeting of CRISPR-Cas9 with a reverse transcriptase to directly edit the . Design choices are made and natural mechanisms of the cell are engaged (or other tools introduced) to ensure that the correct pathway is taken 1) to excise the unedited flap and ligate the edited one and 2) to then edit the DNA of the unedited strand to permanently fix the desired change.
• Prime Editing offers more versatility and flexibility with genome editing especially when compared to Base Editing, and it works in non-dividing cells, albeit with low efficiency.
• Some next steps for Prime Editing are continued refining to improve efficiency and effectiveness, as well as testing in more mammalian and non-dividing (post-mitotic) cell lines. Eventually, we will likely have to come up with creative delivery mechanisms.
• Creating therapies based on Prime Editing (such as for Tay-Sachs) requires adjacent areas of exploration. For example, for the development of gene editing therapy, determining the fraction of edited cells that will result in clinical benefits is critical — genetic diseases differ in the fraction of target cells that must be modified to ameliorate or even cure the disease. For some diseases like certain blood conditions, because the relevant cells can be edited outside the body (ex vivo) and then transplanted back into the patient, this makes editing the relevant cells easier in some respects.
• Further, it requires establishing that the agent can be delivered in an animal model in a manner that reaches the target tissue, understanding to the extent possible any potential side-effects and how to best manage them, optimizing editing efficiency in the target tissue in animals, developing practical ways to manufacture the candidate therapeutic at the high quality and consistency level required for use in humans, and several other key requirements are also crucial to developing new therapies. Addressing these challenges takes years, but is important to maximizing the likelihood that patients benefit from a new therapy (the italicized is a quote from the interview with David R. Liu).

Sources

Beyond the research and articles linked throughout parts 1, 2 and 3, there were a few particularly useful pieces:

This piece from the New England Journal of Medicine was incredibly useful. I went looking on Twitter for ‘prime editing’ until I found strong sources and references. This was the first piece I chose to read for its higher level explanations, and the advantage here is more detailed, accurate and technical breakdown but without going too deep into the weeds.

This piece contains a broader history from the first discovery of CRISPR in 1993, and the gradual uncovering of its function and mechanisms until 2012 (two decades later) when it is well understood enough and first harnessed for human genome editing.

This piece contains a good history and comparison of the detailed differences between original CRISPR-Cas9 (which he called the OG CRISPR which would likely not happen in a scientific article), base editing and prime editing. Because it’s on Medium, the author can add a nice bit of cultural humor and can use metaphors that help us better understand how this works.

Return to pt. 1 or pt. 2.