"DNA is really the genetic treasure of the cell.
DNA is the primary heritable information in
living systems. It is the primary source of
information that we pass down to our children
and even though DNA exists in these complicated
forms, nature has provided us with tools to
access those forms. In the end, like everything
in the natural world, it's all just chemistry.
[crosstalk]
David Liu: "CRISPR began by investigating
how bacteria survive infection from viruses.
We think of bacteria as threats to human health,
as sources of infection, but bacteria are
also prone to being infected. Scientists discovered
that naturally occurring CRISPR is really
a bacterial immune system that uses past encounters
with viruses to program a set of molecular
scissors, now more famously called CRISPR-Cas9,
to cut DNA. It makes what's called a double-stranded
break and in the process destroys the ability
of that DNA to function.
Nicole Gaudelli, David Liu Lab, Harvard University:
"DNA are long stretches of chemical code comprised
of As, Cs, Gs, and Ts, and these four letters
spell out everything that's required for making
hundreds of genes, thousands of proteins.
These different letters base pair or hydrogen
bond with each other to functionally protect
the DNA. When one of those letters is misspelled,
then the instructions, which describe the
proteins that are essential to life, are not
correct. It turns out, a lot of times diseases
are caused simply by a single letter misspelling
in your genetic code."
David Liu: "The pivotal moment in its use
as a genome editing agent came in a series
of papers published at the end of 2012 and
early in 2013 by a variety of scientists,
including Emmanuelle Charpentier, Jennifer
Doudna, George Church, Feng Zhang. These papers
showed that you could program the molecular
scissors to disrupt the function of the genes
that you're cutting. But in most cases, you
may not be interested in simply destroying
the function of a gene, but instead in actually
repairing the gene from a form that causes
the disease back to the healthy form."
Shannon Miller, David Liu Lab, Harvard University:
"CRISPR 2.0 base editing is able to affect
changes that we want in the genome in a really
precise manner."
Nicole Gaudelli: "Base editors are molecular
machines that cleanly convert one base into
another without cutting the DNA."
Shannon Miller: "By binding to DNA and not
creating a double-stranded break, we’re
able to precisely make a single point mutation
without any undesired outcomes that you usually
get with double-stranded break, so you don't
get really large deletions. You don't get
rearrangements."
Nicole Gaudelli: "So CRISPR 1.0 is using scissors
and glue, and CRISPR 2.0 is using a pencil
an eraser."
David Liu: "So to develop base editing, we
began by disabling the CRISPR scissors so
that they no longer could make double stranded
cuts in DNA. Instead, we preserved the ability
of CRISPR to bind to a target DNA sequence.
That's really a very special feature of CRISPR-Cas9
and related CRISPR proteins. Namely, that
they can be programmed with a short piece
of RNA, called a guide RNA, to hone in on
just one DNA sequence of interest and not
be distracted by the billions of other DNA
sequences that might be present in the genome.
"But because DNA is double stranded, when
you change one base of a base pair to something
else, you've actually created mismatched DNA.
So we worried about this disagreement because
the cell in the process of resolving this
disagreement can do so either by changing
the unedited strand, replacing that. Or, what
we didn't want, is it could instead replace
the DNA strand that we just edited, the strand
that we worked so hard to perform the chemistry
on.
"So a very talented postdoc, Alexis Komor,
took on the challenge and started to think
about ways of subverting the cell's DNA repair
response so that it wouldn't undo the work
that our base editors were doing before we
had a chance to make the change permanent.
She imagined that perhaps if we made a small
change to the unedited strand, the one we
wanted replaced, nicking that strand causes
the cell to look at this disagreement and
referee the disagreement in favor of what
we just edited.
"It was one of those moments where you hear
a proposed solution, and you sort of stand
in awe of how beautiful the solution is, how
the natural world occasionally fits together
in just the right way that makes sense and
is simple and easy, rather than hard and complex,
even though the problems you're trying to
tackle at the outset can seem almost insurmountable.
"To be clear, there's a lot of hard work that
still has to be done, but we're incredibly
excited by the fact that there are human genetic
diseases caused by point mutations that might
be treatable, and perhaps even curable, by
using base editors."
