The mutagenic chain reaction: A method for converting heterozygous to
Valentino M. Gantz*, Ethan Bier*
+ Author Affiliations
Section of Cell and Developmental Biology, University of California, San
Diego, La Jolla, CA 92095, USA.
↵*Corresponding author. E-mail: email@example.com (V.M.G.); firstname.lastname@example.org
An organism with a single recessive loss-of-function allele will
typically have a wild-type phenotype while individuals homozygous for
two copies of the allele will display a mutant phenotype. Here, we
develop a method that we refer to as the mutagenic chain reaction (MCR),
which is based on the CRISPR/Cas9 genome editing system for generating
autocatalytic mutations to generate homozygous loss-of-function
mutations. We demonstrate in Drosophila that MCR mutations efficiently
spread from their chromosome of origin to the homologous chromosome
thereby converting heterozygous mutations to homozygosity in the vast
majority of somatic and germline cells. MCR technology should have broad
applications in diverse organisms.
New DNA construct can set off a "mutagenic chain reaction"
Can sweep through an entire population, changing a gene.
by John Timmer - Mar 24, 2015 7:00am AEDT
Flickr user: St3f4n
A technique for editing genes while they reside in intact chromosomes
has been a real breakthrough. Literally. In 2013, Science magazine named
it the runner-up for breakthrough-of-the-year, and its developers won
the 2015 Breakthrough Prize.
The system being honored is called CRISPR/Cas9, and it evolved as a way
for bacteria to destroy viruses using RNA that matched the virus' DNA
sequence. But it's turned out to be remarkably flexible, and the
technique can be retargeted to any gene simply by modifying the RNA.
Researchers are still figuring out new uses for the system, which means
there are papers coming out nearly every week, many of them difficult to
BACTERIAL "IMMUNE SYSTEM" USED TO ENGINEER HUMAN DNA IN HUMAN CELLS
System lets researchers target changes to specific sites in the genome.
That may be precisely why the significance of a paper published last
week wasn't immediately obvious. In it, the authors described a way of
ensuring that if one copy of a gene was modified by CRISPR/Cas9, the
second copy would be—useful, but not revolutionary. What may have been
missed was that this process doesn't stop once those two copies are
modified. Instead, it happens in the next generation as well, and then
the generation after that. In fact, the modified genes could spread
throughout an entire species in a chain reaction, a fact that has raised
ethical and safety concerns about the work.
The CRISPR/Cas9 system is remarkably simple. It relies on RNA molecules
that have a specific format and are able to base pair with a site in the
genome. Cas9 then cuts the DNA at the site where this base pairing
occurs, creating a break in the chromosome. Cells have systems that
attempt to repair these breaks, and these systems attempt to identify
similar-looking sequences to use as a template for repair. So if you
provide the cells with some similar DNA, it will end up being placed at
the site that the RNA first targeted.
This makes it easy to modify the genome. By providing slightly different
DNA to be used in the repair process, you can substitute altered bases,
short deletions, or even entire additional genes, any of which can take
their place within the chromosome. In short, CRISPR/Cas9 lets you put
any DNA you want anywhere in a genome.
It's possible to use this to eliminate genes you're interested in, so
you can study animals that lack that gene. You simply target the gene
with an RNA, and then provide DNA with a deletion of a key part of the
gene. The repair system will use the deletion as part of its template
and copy it into place on the chromosome. It's also possible to mutate a
gene by replacing key parts of it with something else. For example, you
could swap in a copy of the Green Fluorescent protein and ensure that
all of the resulting mutants glow green.
But you still have to breed these mutations the old-fashioned way: you
need to get two organisms that have a copy of the mutant gene, then
breed them together. Mendel then tells us that one-quarter of the
offspring will have mutant copies in both of their chromosomes.
The authors of the new paper found that frustratingly slow. Working in
flies, they designed a system where CRISPR/Cas9 would do all the work
for them. Their DNA repair template was a bit more complicated than a
simple deletion. Instead, it contained the genes needed to get the
CRISPR/Cas9 system to work, along with a guide RNA that targeted a
specific fly gene (in this case, yellow). They surrounded all these
genes with DNA from the yellow gene itself.
Once injected in the fly, the normal yellow gene was disrupted by the
genes for the CRISPR/Cas9 system. Once that happened on one chromosome,
the system could easily perform the same modification on the other
chromosome, making the animal a homozygous yellow mutant.
But the key thing is what happens in the next generation. In these
animals, a normal copy of the yellow gene comes in from the next parent.
But the CRISPR/Cas9 cassette immediately converts that, too, resulting
in offspring that are all yellow. Well, not all; but the authors found
that the construct was 97 percent effective at converting the next
generation. In fact, there's nothing to stop this system from invading
an entire population, continuing to convert generation after generation
until everything carries the modification.
It's a bit like the futurists' fears of a self-perpetuating "grey goo,"
just played out with yellow-colored flies. (For those of you with a
biology background, this will also sound a lot like an engineered homing
Fortunately, the researchers were conscious of the issues: "we are also
keenly aware of the substantial risks associated with this highly
invasive method since the failure to take stringent precautions could
lead to the unintentional release of [modified] organisms into the
environment." The flies were bred behind three layers of containment in
a locked facility. The containers were put straight into the freezer to
kill the flies if they were no longer needed. Any manipulations of the
flies were performed while they were anesthetized in a Biosafety Level 2
Still, there are further precautions that could be taken. The report
cites a draft manuscript, hosted on the bioRxiv, that describes a
similar system in yeast. In this case, however, only the targeting RNA
is inserted into the targeted gene—the rest of the CRISPR/Cas9 system
has to be provided separately for anything to happen. One of the authors
of this manuscript, the synthetic biologist George Church, told a
Science reporter that he felt the fly work should never have been
published because the technology was too dangerous.
Why do the work at all if it's so risky? Because, properly controlled,
there could be some amazing benefits. Imagine using it to quickly breed
traits from non-agricultural plants (drought or pest resistance, for
example) into important food crops. Or converting the entire population
of a dangerous pathogen into one lacking virulence genes. Or releasing a
few mosquitos, allowing them to breed, and creating a population that's
incapable of supporting malarial parasite growth. All of these are very
real possibilities enabled by the technology.
But there's also a very real risk of a giant, uncontrolled experiment if
any of these DNA constructs made it into a wild population. And the
developments come at a time where several researchers (including Church)
have suggested it's time to lay out some formal guidelines for future
research in this area, both for synthetic biology and for human genome
modifications. The authors of the fly paper cite two of these
editorials, while Science and Nature have run editorials urging that we
avoid editing the human germline.
The authors of the fly paper suggest looking to the Asilomar agreement,
which was forged by leading biologists who were leaders in the
development of recombinant DNA. That created a voluntary yet successful
moratorium on the work until safety issues could be examined. We may be
forced to see whether this sort of voluntary agreement would hold in an
era of intense competition. Similar concerns were voiced about work
involving flu viruses, but research continued until the federal
government announced a halt to funding for this research.