Can we cure genetic diseases by rewriting DNA David R. Liu
The most important gift
your mother and father ever gave you
was the two sets
of three billion letters of DNA
that make up your genome.
But like anything
with three billion components,
that gift is fragile.
Sunlight, smoking, unhealthy eating,
even spontaneous mistakes
made by your cells,
all cause changes to your genome.
The most common kind of change in DNA
is the simple swap of one letter,
or base, such as C,
with a different letter,
such as T, G or A.
In any day, the cells in your body
will collectively accumulate
billions of these single-letter swaps,
which are also called “point mutations.”
Now, most of these
point mutations are harmless.
But every now and then,
a point mutation disrupts
an important capability in a cell
or causes a cell to misbehave
in harmful ways.
If that mutation were inherited
from your parents
or occurred early enough
in your development,
then the result would be
that many or all of your cells
contain this harmful mutation.
And then you would be one
of hundreds of millions of people
with a genetic disease,
such as sickle cell anemia or progeria
or muscular dystrophy
or Tay-Sachs disease.
Grievous genetic diseases
caused by point mutations
are especially frustrating,
because we often know
the exact single-letter change
that causes the disease
and, in theory, could cure the disease.
Millions suffer from sickle cell anemia
because they have
a single A to T point mutations
in both copies of their hemoglobin gene.
And children with progeria
are born with a T
at a single position in their genome
where you have a C,
with the devastating consequence
that these wonderful, bright kids
age very rapidly and pass away
by about age 14.
Throughout the history of medicine,
we have not had a way
to efficiently correct point mutations
in living systems,
to change that disease-causing
T back into a C.
Perhaps until now.
Because my laboratory recently succeeded
in developing such a capability,
which we call “base editing.”
The story of how we developed base editing
actually begins three billion years ago.
We think of bacteria
as sources of infection,
but bacteria themselves are also
prone to being infected,
in particular, by viruses.
So about three billion years ago,
bacteria evolved a defense mechanism
to fight viral infection.
That defense mechanism
is now better known as CRISPR.
And the warhead in CRISPR
is this purple protein
that acts like molecular
scissors to cut DNA,
breaking the double helix into two pieces.
If CRISPR couldn’t distinguish
between bacterial and viral DNA,
it wouldn’t be a very useful
defense system.
But the most amazing feature of CRISPR
is that the scissors can be
programmed to search for,
bind to and cut
only a specific DNA sequence.
So when a bacterium encounters
a virus for the first time,
it can store a small snippet
of that virus’s DNA
for use as a program
to direct the CRISPR scissors
to cut that viral DNA sequence
during a future infection.
Cutting a virus’s DNA messes up
the function of the cut viral gene,
and therefore disrupts
the virus’s life cycle.
Remarkable researchers including
Emmanuelle Charpentier, George Church,
Jennifer Doudna and Feng Zhang
showed six years ago how CRISPR scissors
could be programmed
to cut DNA sequences of our choosing,
including sequences in your genome,
instead of the viral DNA sequences
chosen by bacteria.
But the outcomes are actually similar.
Cutting a DNA sequence in your genome
also disrupts the function
of the cut gene, typically,
by causing the insertion and deletion
of random mixtures of DNA letters
at the cut site.
Now, disrupting genes can be very
useful for some applications.
But for most point mutations
that cause genetic diseases,
simply cutting the already-mutated gene
won’t benefit patients,
because the function of the mutated gene
needs to be restored,
not further disrupted.
So cutting this
already-mutated hemoglobin gene
that causes sickle cell anemia
won’t restore the ability of patients
to make healthy red blood cells.
And while we can sometimes introduce
new DNA sequences into cells
to replace the DNA sequences
surrounding a cut site,
that process, unfortunately, doesn’t work
in most types of cells,
and the disrupted gene outcomes
still predominate.
Like many scientists,
I’ve dreamed of a future
in which we might be able to treat
or maybe even cure
human genetic diseases.
But I saw the lack of a way
to fix point mutations,
which cause most human genetic diseases,
as a major problem standing in the way.
Being a chemist, I began
working with my students
to develop ways on performing chemistry
directly on an individual DNA base,
to truly fix, rather than disrupt,
the mutations that cause genetic diseases.
The results of our efforts
are molecular machines
called “base editors.”
Base editors use the programmable
searching mechanism of CRISPR scissors,
but instead of cutting the DNA,
they directly convert
one base to another base
without disrupting the rest of the gene.
So if you think of naturally occurring
CRISPR proteins as molecular scissors,
you can think of base editors as pencils,
capable of directly rewriting
one DNA letter into another
by actually rearranging
the atoms of one DNA base
to instead become a different base.
Now, base editors don’t exist in nature.
In fact, we engineered
the first base editor, shown here,
from three separate proteins
that don’t even come
from the same organism.
We started by taking CRISPR scissors
and disabling the ability to cut DNA
while retaining its ability to search for
and bind a target DNA sequence
in a programmed manner.
To those disabled CRISPR
scissors, shown in blue,
we attached a second protein in red,
which performs a chemical reaction
on the DNA base C,
converting it into a base
that behaves like T.
Third, we had to attach
to the first two proteins
the protein shown in purple,
which protects the edited base
from being removed by the cell.
The net result is an engineered
three-part protein
that for the first time
allows us to convert Cs into Ts
at specified locations in the genome.
But even at this point,
our work was only half done.
Because in order to be stable in cells,
the two strands of a DNA double helix
have to form base pairs.
And because C only pairs with G,
and T only pairs with A,
simply changing a C to a T
on one DNA strand creates a mismatch,
a disagreement between the two DNA strands
that the cell has to resolve
by deciding which strand to replace.
We realized that we could further engineer
this three-part protein
to flag the nonedited strand
as the one to be replaced
by nicking that strand.
This little nick tricks the cell
into replacing the nonedited G with an A
as it remakes the nicked strand,
thereby completing the conversion
of what used to be a C-G base pair
into a stable T-A base pair.
After several years of hard work
led by a former post doc
in the lab, Alexis Komor,
we succeeded in developing
this first class of base editor,
which converts Cs into Ts and Gs into As
at targeted positions of our choosing.
Among the more than 35,000 known
disease-associated point mutations,
the two kinds of mutations
that this first base editor can reverse
collectively account for about 14 percent
or 5,000 or so pathogenic point mutations.
But correcting the largest fraction
of disease-causing point mutations
would require developing
a second class of base editor,
one that could convert
As into Gs or Ts into Cs.
Led by Nicole Gaudelli,
a former post doc in the lab,
we set out to develop
this second class of base editor,
which, in theory, could correct up to
almost half of pathogenic point mutations,
including that mutation that causes
the rapid-aging disease progeria.
We realized that we could
borrow, once again,
the targeting mechanism of CRISPR scissors
to bring the new base editor
to the right site in a genome.
But we quickly encountered
an incredible problem;
namely, there is no protein
that’s known to convert
A into G or T into C
in DNA.
Faced with such a serious stumbling block,
most students would probably
look for another project,
if not another research advisor.
(Laughter)
But Nicole agreed to proceed with a plan
that seemed wildly ambitious at the time.
Given the absence
of a naturally occurring protein
that performs the necessary chemistry,
we decided we would evolve
our own protein in the laboratory
to convert A into a base
that behaves like G,
starting from a protein
that performs related chemistry on RNA.
We set up a Darwinian
survival-of-the-fittest selection system
that explored tens of millions
of protein variants
and only allowed those rare variants
that could perform the necessary
chemistry to survive.
We ended up with a protein shown here,
the first that can convert A in DNA
into a base that resembles G.
And when we attached that protein
to the disabled CRISPR
scissors, shown in blue,
we produced the second base editor,
which converts As into Gs,
and then uses the same
strand-nicking strategy
that we used in the first base editor
to trick the cell into replacing
the nonedited T with a C
as it remakes that nicked strand,
thereby completing the conversion
of an A-T base pair to a G-C base pair.
(Applause)
Thank you.
(Applause)
As an academic scientist in the US,
I’m not used to being
interrupted by applause.
(Laughter)
We developed these
first two classes of base editors
only three years ago
and one and a half years ago.
But even in that short time,
base editing has become widely used
by the biomedical research community.
Base editors have been sent
more than 6,000 times
at the request of more than
1,000 researchers around the globe.
A hundred scientific research papers
have been published already,
using base editors in organisms
ranging from bacteria
to plants to mice to primates.
While base editors are too new
to have already entered
human clinical trials,
scientists have succeeded in achieving
a critical milestone towards that goal
by using base editors in animals
to correct point mutations
that cause human genetic diseases.
For example,
a collaborative team of scientists
led by Luke Koblan and Jon Levy,
two additional students in my lab,
recently used a virus to deliver
that second base editor
into a mouse with progeria,
changing that disease-causing
T back into a C
and reversing its consequences
at the DNA, RNA and protein levels.
Base editors have also
been used in animals
to reverse the consequence of tyrosinemia,
beta thalassemia, muscular dystrophy,
phenylketonuria, a congenital deafness
and a type of cardiovascular disease –
in each case, by directly
correcting a point mutation
that causes or contributes to the disease.
In plants, base editors have been used
to introduce individual
single DNA letter changes
that could lead to better crops.
And biologists have used base editors
to probe the role of individual letters
in genes associated
with diseases such as cancer.
Two companies I cofounded,
Beam Therapeutics and Pairwise Plants,
are using base editing
to treat human genetic diseases
and to improve agriculture.
All of these applications of base editing
have taken place in less
than the past three years:
on the historical timescale of science,
the blink of an eye.
Additional work lies ahead
before base editing can realize
its full potential
to improve the lives of patients
with genetic diseases.
While many of these diseases
are thought to be treatable
by correcting the underlying mutation
in even a modest fraction
of cells in an organ,
delivering molecular machines
like base editors
into cells in a human being
can be challenging.
Co-opting nature’s viruses
to deliver base editors
instead of the molecules
that give you a cold
is one of several promising
delivery strategies
that’s been successfully used.
Continuing to develop
new molecular machines
that can make all of the remaining ways
to convert one base pair
to another base pair
and that minimize unwanted editing
at off-target locations in cells
is very important.
And engaging with other scientists,
doctors, ethicists and governments
to maximize the likelihood
that base editing is applied thoughtfully,
safely and ethically,
remains a critical obligation.
These challenges notwithstanding,
if you had told me
even just five years ago
that researchers around the globe
would be using laboratory-evolved
molecular machines
to directly convert
an individual base pair
to another base pair
at a specified location
in the human genome
efficiently and with a minimum
of other outcomes,
I would have asked you,
“What science-fiction novel
are you reading?”
Thanks to a relentlessly dedicated
group of students
who were creative enough to engineer
what we could design ourselves
and brave enough
to evolve what we couldn’t,
base editing has begun to transform
that science-fiction-like aspiration
into an exciting new reality,
one in which the most important gift
we give our children
may not only be
three billion letters of DNA,
but also the means to protect
and repair them.
Thank you.
(Applause)
Thank you.