
Real-time dynamics of CRISPR visualized by high-speed atomic force microscopy - kjhughes
https://www.nature.com/articles/s41467-017-01466-8
======
kjhughes
Here's a quick peek at the visualization tweeted by one of the authors:

[https://twitter.com/hnisimasu/status/928933260159197184](https://twitter.com/hnisimasu/status/928933260159197184)

And an article in The Atlantic that some may find more approachable than the
original Nature Communications publication:

[https://www.theatlantic.com/science/archive/2017/11/crispr-v...](https://www.theatlantic.com/science/archive/2017/11/crispr-
video-real-time/545603/)

~~~
chiefalchemist
There was also this in The Atlantic that overviews a different - perhaps
somewhat conflicting? - theory on genetics

[https://www.theatlantic.com/science/archive/2017/06/its-
like...](https://www.theatlantic.com/science/archive/2017/06/its-like-all-
connected-man/530532/)

------
jfarlow
Pretty cool visualization. Single molecule work like that is challenging to do
properly - but when done properly gives a kind of visual proof to a hypothesis
that is hard to controvert. Wild-type Cas9 cuts genomic DNA, and when it does,
the strand can go flying - with repair a possibility rather than an
inevitability.

Cas9 (and similar proteins) have 2 basic functions:

1) to bind to specific DNA (by means of complementing a specifically-bound
RNA)

2) to cut the DNA that is has bound.

Only one of these functions is actually desirable or unique to Cas9 - that of
binding to a sequence-specific part of DNA. The 'cutting' function is how the
CRISPR system naturally destroys foreign DNA in Cas9's normal environment, but
is generally not desirable in these synthetic biology or therapeutic
applications. For genetic engineering purposes we really have better tools
than Cas9 to cut, modify, or otherwise affect DNA - but Cas9 is clearly a
fantastic tool to _localize_ to particular sequences. That this paper so
clearly demonstrates the terrifying damage that cleavage can do to genomic DNA
only reinforces that Cas9 is a useful scaffold upon which to engineer further
tools, but in its wild-type form is kind of scary.

Recent papers from the Liu lab at Harvard [1] (among many others) show how you
can edit DNA using a modified Cas9 that has been modified to _not_ have that
second cutting function, while retaining the ability to bind to a specific DNA
sequence. The synthetic "BaseEditor" protein has been further modified to have
more complicated DNA _modification_ capabilities, rather than whole-sale
slicing of DNA [2]. Though this designed protein with Cas9 at its core is
complicated, it should not cleave and let dangle genomic DNA as we see it here
visualized. I'd be curious to see how the BaseEditors behave under a similar
visualization.

[1] Kim et al. 2017:
[https://www.nature.com/articles/nbt.3803](https://www.nature.com/articles/nbt.3803)

[2] BaseEditor v4:
[https://serotiny.bio/notes/proteins/sabe4gam/](https://serotiny.bio/notes/proteins/sabe4gam/)

~~~
nonbel
>"Wild-type Cas9 cuts genomic DNA, and when it does, the strand can go flying
- with repair a possibility rather than an inevitability."

"Possibility" is too strong a term. Afaict, the best you can say about repair
is that it is a "less than common occurrence," and even that may be too much.

>"Recent papers from the Liu lab at Harvard [1] (among many others) show how
you can edit DNA using a modified Cas9 that has been modified to not have that
second cutting function, while retaining the ability to bind to a specific DNA
sequence."

Sorry but I could not find any comment/data about toxicity in this paper. This
is basic info everyone wants to know: What percent of cells exposed to this
stuff die? Maybe I missed it?

~~~
jfarlow
I understand your hypothesis that Cas9 only kills the cells that do not have
the particular mutation and itself is a negative selection against the
undesired genetic sequence. I do not believe the basic numbers and statistics
of the rate of toxicity, size of genome, precision of mutation, etc. work out
to permit that hypothesis, but it is a curious thought. As a hypothesis it is
straightforward to test with a FACS-sorted population of cells expressing a
fluorescent Cas9: does the fluorescent population die, or does the fluorescent
population survive and contain the expected genetic mutation.

That experiment has been done and is actually written up here as a protocol to
more effectively using Cas9. The paper clearly refutes the above hypothesis
and demonstrates that modifications by Cas9 are generally proportional to the
amount of expressed Cas9 up to the point of toxicity [1]. In it they tag Cas9
with a fluorophore, express the construct in cells and sort for those cells
that express Cas9 (separating them from cells that do not), and binning the
cells based on the level of Cas9 expression. Given your hypothesis, those
fluorescent cells should die and not have the desired mutations, while the
non-fluorescent cells will be enriched for particular mutations not found in
the fluorescent cells. Some toxicity is shown at very high levels of Cas9
expression, but the on-target edits occur in cells expressing Cas9, and those
cells live for a number of passages. See Figure 3 in the paper below:

> Cell population 1 displays GFP fluorescence intensities similar to those of
> nontransfected cells (autofluorescence level), whereas populations 2–6
> display specific GFP levels ranging from low to high (varying ~500-fold).
> (e) 20,000 cells were isolated from each of populations 1–6 in (d) and
> cultured for 6 d, after which IDAA was used to determine indels at the KRAS
> locus in an aliquot of the cells. Note that editing levels increase with
> increasing GFP fluorescence level at the time of FACS and that almost
> complete editing was obtained at submaximal fluorescence levels... [T]he
> total number of live cells in each population analyzed in (e) was determined
> by trypan blue exclusion and expressed as % of population 1.

[1]
[https://www.nature.com/articles/nprot.2016.165](https://www.nature.com/articles/nprot.2016.165)

Given the above result, a _significant_ amount of research is being done
_right now_ to preserve the useful properties of Cas9 - being able to home in
on a particular genetic sequence - while engineering the protein to minimize
and even prevent those deleterious cuts do DNA. And that research is finding
steady success.

~~~
nonbel
>"I understand your hypothesis that Cas9 only kills the cells that do not have
the particular mutation"

The hypothesis is that it is more likely to kill those cells than the others,
and does so at a high enough rate to select for cells missing the target site.
Other things such as NHEJ/HDR happen too.

Anyway, the technique in the paper you linked is not supposed to be causing
double strand breaks, which for a long time before CRISPR/Cas-9 were thought
to result in Death >> NHEJ >> HDR. This is a totally different scenario, but
it is quite odd they do not mention toxicity at all since that is a major
concern of these technologies.

I'll check the Lonowski et al paper and get back to you.

EDIT:

> "Given your hypothesis, those fluorescent cells should die and not have the
> desired mutations, while the non-fluorescent cells will be enriched for
> particular mutations not found in the fluorescent cells"

Perhaps there is a typo here but this is exactly the opposite of what my
hypothesis predicts. The surviving cells should contain cas-9 _and_ the
mutation. Cas-9 damages the DNA of some cells less because the the mutation
was already there. The cells with no cas-9 are noise because they were never
treated.

~~~
jfarlow
> Cas-9 damages the DNA of some cells less because the the mutation was
> already there. The cells with no cas-9 are noise because they were never
> treated.

If the mutations were already there, then control in figure 7 in Lonowski et
al. would not contain 0% "on-target" mutations. The presence of Cas9 is
clearly _causing_ a genetic change that is not already present in the
population, and those cells that are changed do not all die. It is clearly not
the case that the cells with the desired mutations completely overlapped with
the cells transfected.

~~~
nonbel
>"Although exhibiting background fluorescence, population 1 expresses some
nuclease, as evidenced by a low level of editing. [...] The ‘shoulder’
indicated by an asterisk is not an indel, as it is also present in the control
sample; it is probably due to incomplete 3´ A nucleotide addition to the IDAA
amplicon (see Table 3, Step 21)."

So they do see mutations in the control cells but decided to call it some kind
of artefact. BTW, I also see a less shoulder in the left (on-target) site of
the same figure.

The point is that these mutants pre-exist in low frequencies of 1/100 to
1/100,000 cells (depends on cell type, environment, etc), almost regardless of
the target site. If you look at enough cells you are guaranteed to find one
with an indel at any site. In those cases, just as cas-9 doesn't just keep re-
editing the dna after the target sequence has been cut, the cells are
relatively "immune" to it.

So for figure 7 they started with 50k cells, the questions are:

1) How many of those cells already contained a mutation at the target site?

2) What was the growth curve of the cells after treatment?

3) What is the division rate of these cells under these conditions?

4) What is the death rate of these cells under these conditions?

5) How many cells were there in the end for final analysis (sequencing,
whatever)?

------
amelius
Does anyone know if there are also AFM videos of standard biological
processes, e.g. molecular machines such as kinesin [1]?

EDIT: Found one here: [2].

[1]
[https://en.wikipedia.org/wiki/Kinesin](https://en.wikipedia.org/wiki/Kinesin)

[2]
[https://www.youtube.com/watch?v=1jt3AM5Kfqw](https://www.youtube.com/watch?v=1jt3AM5Kfqw)

------
magnat
It's surprisingly slow process. Tens of seconds seem like ages in molecular
physics. One could think an interaction between molecules should happen on a
sub-nanosecond scale.

~~~
positr0n
I was shocked the video was 20 seconds in real time as well. I wonder if there
is a "numbers every programmer should know"[0] analog for chemical biologists.

[0]:
[https://gist.github.com/jboner/2841832](https://gist.github.com/jboner/2841832)

