Wow, this is CRISPR.
Are we gonna succeed?
There's always a chance that you can blow it.
Let's see what happens.
So Roland, everyone says CRISPR's really easy to do
and i was interviewing a very prominent researcher
the other day, who said to me:
"Any idiot can do CRISPR."
Is that true?
Uh, I'm not sure about any idiot but it is easy
and we're going to find out.
So what we want to do is we want to knock out
a gene in a human cell line.
Yeah, I chose a gene that putatively is
involved with the immune response to Dengue virus.
And the reason I chose it
is because there's a theory that
if you've had Dengue virus
and you have an antibody to it
and then get infected with Zika-
it helps the Zika virus copy itself
which might be responsible
for the damage that we're seeing in babies.
So, you and I are going to solve that problem right?
hopefully, I mean...
What are the basic steps that we need to do
to get started?
First, we'll have to define the genomic region
that we want to cut.
Then we take that information and put it into a
design software, in a CRISPR design software.
And the third step is ordering
the oligo nucleotides for the cloning of the guide RNAs.
Then you have to order a plasmid.
What's the name of our plasmid?
It's lentiCRISPR v2.
And we're going to put in here our
20 nucleotide sequence that's going to guide us to
the portion of the gene that we're gonna knock out.
Exactly.
So we've got the oligos
and we've got the plasmids ready to go.
Now we're going to pipette the...
Now we're gonna pipette everything...
We're going to pipette it all together. Right.
OK let's do it.
So, first of all we take a tube.
This tube is sterile.
No, see like you already have the problem:
we don't know what a tube is, right?
So this is like actually a microcentrifuge tube right?
It's at 1.5 milliliter microcentrifuge tube.
This is where we will set up our reaction.
So the components we will need is:
the plasmid, right.
The enzyme. Got it.
The enzyme only works in a specific environment,
so we'll also need the buffer, the correct buffer.
And we also make it like a feasible volume,
so we'll add water to it.
Go down.
Push the bottom down and up.
Just one time?
So, um, so, here's the deal, hold on Jon like
can I take the pipette?
You were way too fast so you had bubbles in there.
So, let's try it again.
So I already ruined the experiment?
Basically, by not knowing how to pipette you're already
like on a bad path right?
Now don't, not failed necessarily,
it's very robust but its sub-optimal.
Alright, here we go again.
That looks good, OK.
Alright. Hooh.
Very nice.
Easy peasy.
I didn't know it was so much fun to do CRISPR.
It is.
And now let go of the...
Yes, that was good.
That was proper pipetting.
Oops, yeah I think now you kind of like
you failed a little bit probably because you put it...
See how much liquid there's still left?
Yeah, should be fine you know?
Let's keep going.
And now you vortex again.
It's a little nerve-racking.
I don't want make a mistake so...
Woo, I'm vortexing
You're totally vortexing right now.
And then you spin it real quickly.
[spinning sound]
That's it. That's it.
It goes on 37. Let's go over there.
Put this in the water bath.
I don't think we need to videotape that one but...
In the beginning I said: "Is it it really easy?"
You know, " Can any idiot do this?" "Yes," but then like
I got the sense that like there's some pride that, no,
you've got to really be kind of skilled do it.
I think there is a certain skill level
It's very necessary right?
Like, if you take a random person from the street
and say knockout cd32 in thb cells
in like mammalian cells- there is no way
this person would do it, not in a hundred years.
Not gonna happen.
it's really easy for people
who already know how to do what?
To do molecular biology.
We just discussed, pretty much everyone
knows about DNA.
Go to RNA and most people are like, ahhh
not so sure, right?
Mention oligos, plasmids, cloning and so on,
You will lose everybody. Yeah.