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[dupe] CRISPR-Cas9 gene editing causes lots of mutations (dw.com)
96 points by pmoriarty on July 19, 2018 | hide | past | web | favorite | 47 comments

Cas9 does not edit DNA, it cleaves DNA. What happens afterwards is up to the cell. The hype machine has ignored this fact.

One of my favorite George Church quotes: "CRISPR: some call it genome editing. I call it genome vandalism".

Base Editors can actually edit DNA, but only single bases at a time. http://www.sciencemag.org/news/2017/10/novel-crispr-derived-...

There's a big prize waiting for the person who can harness DNA repair pathways in conjunction with Cas9 to make precise, multi-base DNA edits. Lots of folks are working on that now.

Does it cleave a whole segment off (two point cut), or just a single cut?

Single cut.

Sometimes I think these gene editing techniques are more like having a fantastic hex editor... but with a 5GB binary.

I think that if you wanted to reframe it this way, it's more like like editing a 800MB binary with sed/regexes (with some degree of random side effects injected sometimes)

And no way to really test it without just running it in production!

Move fast and break (biological) stuff

Really curious how far are we from being able to simulate the human body's response to drugs/modification.

We are so ridiculously far that it's not really worth thinking about. We're still at the stage of simulating how molecules interact with each other. To work our way up even to the single-cell organism level would be a historic human achievement.

To simulate an entire human body is computationally mindboggling. The number of cells are in the tens of trillions and we'd need to simulate their responses, not just to the drugs we care about, but doing so while "operating" (feeding, sleeping, etc.) the simulated body.

I have no experience in this area, but. Once you get the molecules and their interactions down wouldn't that make all other types of cells easier to produce?

And at a certain point couldn't you just feed the simulation DNA and watch it grow?

It takes supercomputers days to simulate milliseconds of interactions between a million atoms using classical mechanics (ie Newton's Laws, basically a ball and spring model so not even accounting for quantum effects except through very rough approximations in the spring constants). We don't even have an accurate model for water yet.

The oldest code we have is probably ~65 years.

Our genomes are ~1.2B to 3.5B (sexual reproduction and life at all, respectively) years old.

Now imagine trying to patch that codebase in production...

And sometimes you end up with two files instead of one.

Or two flies instead of one.

This is not a bad analogy. I got my start in computers but moved to biology and spent a long time thinking of gene therapy as a hex editor for patching genomes.

The reality is so much more complicated, though, that even good analogies don't provide us with any path forward in terms of actual changes to make.

What part is more complicated? Ie, is it the editing process is imprecise? Perhaps the editing process is like using sed (as mentioned by other people)?

In simple terms, at least.

You mean the editing? The editing depends on fairly complicated processes that originally evolved (to the extent that one can impute function) to repair drastic problems.

The complicated bit comes in knowing what to change. There are some diseases in which the entire disease is caused by a single mutation and changing that one mutation in the germ line would be sufficient to correct for the disease. However, most situations involve complex interactions with many genes in a way that making a single or small simple change would have many side effects, or cause other serious problems.

It's like editing a 500Gb text file on disk by trying to flip the magnetic bits with a tiny magnet

Wait...is that not how HDD's work already?

Except the hex (or binary) editor isn't quite perfect yet. Improving this is obviously very important for gene editing.

I think the problem is also, that the code which was the base for the binary is also pretty crappy legacy code without encapaulation etc.

Or built with an optimizing compiler. Point being, just like with typical binaries, you can't easily read what the program will be doing; it's not apparent in the structure of data.

and the 5GB binary has been aggressively obfuscated, with dead code, duplications, random jumps and weird coupling all over the place - and you don't really have a hex editor, just regexes...

Not entirely unexpected to see stuff like this come up. We're still in the early days of what gene editing is and will become. Keeping my fingers crossed for its future.

tl;dr: (1) The paper's main finding has been known by the DNA repair community, (2) relying on DNA double-strand breaks for genome editing has been known to be non-ideal in the long-term, and (3) current ex vivo therapies are unaffected since we can screen cells after editing.

The root cause here is the DNA double-strand break induced by Cas9, and the ensuing DNA repair processes that occassionally produces large deletions (the paper reports two thirds or 32/48 were less than 50 bp and 21% or 10/48 were greater than 250 bp). This has been largely known by the DNA repair community for 20+ years.

Currently, our most powerful genome editing techniques rely on CRISPR's ability to induce double-strand breaks, such as HDR (for inserting designed sequences, used in gene drives for instance) and NHEJ (for knocking out genes, used in all current early-phase therapies under development). However, a double-strand break is among the most traumatic experiences a cell's genome can experience. We're currently taking advantage of the cell's freak-out attempts to repair its DNA for genome editing purposes, but the process is noisy and produces highly variable and stochastic outcomes. HDR, for instance, has a baseline efficiency of less than 20% in many cell types and conditions of therapeutic interest. Many papers have shown dramatically increased HDR rates at the expense of altering the cell's DNA repair pathways, but these were never going to be humanity's dominant therapeutic approach -- messing with DNA repair is dangerous and closely linked to cancer.

The holy grail is a genome-editing tool that is highly precise, with no off-target effects (edits only exactly in the genome where we want) and highly precise on-target effects (results in the same change everytime we apply it) and flexilbe (many designable changes). The messiness of cellular DNA repair is a major challenge to the second goal. The scientific community has recognized this and has been working on CRISPR-related genome-editing technologies that rely less or not at all on inducing double-strand breaks, thereby skipping the messiest part of the genome-editing process.

One example is base editing, where the endonuclease domains of Cas9 are disabled (called dCas9, sometimes explained as "dead" Cas9), and alternative enzymes that transform nucleotides (such as C->T) are attached to dCas9. In this setup, dCas9 provides the "homing" targeting while the attached enzyme enacts the editing without inducing a DSB. As it stands now, base editing is very exciting, but further work remains to develop base editors for all possible nucleotide transformations (a -> b for all a != b in {A, C, G, T}); it's not as flexible as HDR.

I do want to note another therapeutic domain of ex vivo therapies, where the messiness of DSB-associated genome editing strategies is much less of an issue, since cells can be screened after editing in vitro to control which genotypes we're inserting back into the patient.

>"current ex vivo therapies are unaffected since we can screen cells after editing."

If this is so easy then why is it not standard practice? From the paper:

>"Results reported here also illustrate a need to thoroughly examine the genome when editing is conducted ex vivo. As genetic damage is frequent, extensive and undetectable by the short-range PCR assays that are commonly used, comprehensive genomic analysis is warranted to identify cells with normal genomes before patient administration." https://www.nature.com/articles/Nbt.4192

How much more expensive and time consuming will this procedure become if you require "comprehensive genomic analysis"? The merit of crispr/cas9 over other techniques was supposed to be how cheap and quick it was.

How many gene editors does it take to change a lightbulb?

Only one, but you might also get inflammation, organ failure, virus infection, cancer or autoimmune disease.

As a software engineer super interested in genome editing, are there any software out there that will educate me through the process?

Nearly all the software are tools for designing CRISPR gRNAs, not really educational software. There are videos and books for learning more about the whole thing though.

Base editors can make all 12 conversions as of 4ish months ago I think.

Every time I read about gene editing, I think of the short story Sisters by Greg Bear: https://www.baen.com/Chapters/ERBAEN0036/ERBAEN0036___1.htm

Isn't this the basis for the movie "I Am Legend"?

In all seriousness, how likely will the gene mutations from people self experimenting lead to a contagion? There are many CRISPR kits that people can buy to do their own experimentation.

I suspect you're much more likely to give yourself cancer than cause an I am Legend-style mutation.

Probably so, but I don't have any statistical data to form an opinion. Does anyone track genetic incidents / blunders at a global scale?

>CRISPR-Cas9 gene editing causes lots of mutations

This is still a very controversial statement, and far from definitively proven. Yes, even with a nature paper.

I also take issues with the phrase "lots". So far this effect has been observed only locally.

I believe the result, and I think it needs broader investigation but a summary like "gene-editing causes lots of mutations" is not accurate.

I don't think this is a controversial statement at all. Any CRISPR-Cas9 paper published will at least discuss the prospect of undesired mutations; this is a well-known concern.

You may be interested in my comment from the last time this journal article was posted to HN.


Speaking as a biologist. Among my colleagues, it is still very controversial.

It should be noted that "controversial" scientifically means something different than in the general public. What I mean is that we see and believe the current observations but are still waiting to fully understand the extent and reproducibility.

> Speaking as a biologist. Among my colleagues, it is still very controversial.

I'm also a biologist.

I guess I don't understand where your concern arises. I and others I know have used CRISPR-Cas9 to introduce arbitrary DNA mutations at loci of interest. We see many different types of lesions, ranging in size.

I shouldn't have brought credentials into this, its bad form on my part.

But anyway, I was just relating what I've heard from colleagues and my dis-taste for the articles definitive title.

Not quite true. There is a decent amount of literature describing off-target cleavage events as well.

Yeah I fear like all this will do is give rise to some false conspiracy theories about CRISPR.

There is a wide range of literature that supports the claim. Some of it is garbage but there's enough evidence that any scientist should be suspicious of claims there isn't.

>Some of it is garbage

This is why it is controversial.

I said some of it is garbage. Not all. Making claims in this area is fraught with incompetence and lying, so my warning is to skilled readers who need to be reminded of the value of skepticism.

I agree.

I am taking issue with the articles title "CRISPR-Cas9 gene editing causes lots of mutations"

From the current literature, there have been bad and good examples of this, but it is still a field in flux. We do not know conclusively what the effect is, how large, and how local. This makes blanket statements like "CRISPR-Cas9 gene editing causes lots of mutations" controversial.

Yeah. It seems a bit like saying "use of cars causes lots of deaths". As we learn how to drive that should hopefully become less of a problem.

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