by Heidi Ledford
05 April 2017
Scientists face tough decisions
when the latest gene-editing
don't match up with the results
of other techniques.
It seemed like the
Jason Sheltzer, a
cancer biologist at Cold Spring Harbor Laboratory in New York, was
on the hunt for genes involved in tumor growth.
He and his
colleagues planned to disable genes using
the popular gene-editing tool CRISPR-Cas9, then look for changes
that reduced the rate at which cancer cells multiply. But they
needed a control gene that would yield that same effect.
suggested that the gene
MELK was ideal:
there was ample
evidence that it is important in cancer-cell proliferation, and
clinical trials are under way to test drugs that inhibit the MELK
But disabling the gene using
CRISPR-Cas9 yielded no effect.
"That threw a
monkey wrench into our experiments," says Sheltzer. "It brought
everything to a halt."
With that result,
Sheltzer and his team joined an expanding club of laboratories that
have been forced to re-evaluate and repeat experiments, as the
spread of CRISPR-Cas9 uncovers potential errors in data collected
using older techniques.
On 3 April, Sheltzer's team presented the
findings at the American Association for Cancer Research annual
meeting in Washington DC.
The results have also been published in
the journal eLife. 1
whole lot of work to be done, just basically repeating the same
screens that people had done" with other methods, says Michael
Bassik, a molecular biologist at Stanford University in
"And, I think
it's fair to say, to get much better data."
Scale of the problem
Nathan Lawson, a
molecular biologist at the University of Massachusetts Medical
School in Worcester, was one of the first to systematically
characterize the problem.
In 2015, he and his colleagues reported
their efforts to compare results from two methods in
knocking out genes using a gene-editing technique called zinc finger
nucleases, and reducing gene expression using molecular tools called morpholinos.
They found that half of the 20 genes they tested
yielded different results. 2
An additional trawl
through genetic databases and
the morpholino literature revealed
that 80% of results from published morpholino experiments were not
reproducible in genetic mutants. 2
researchers said they welcomed the paper because it forced the
community to confront a problem that had only been noted
Others were not so happy.
"I got some people who told
me I ruined the field," says Lawson.
have cropped up in other organisms. In the model plant
Arabidopsis thaliana, the use of CRISPR-Cas9 showed that a
protein previously thought to mediate the effects of the plant
hormone auxin does not have that function. 3
In fruit flies and
human cells, large screening studies have turned up widespread
discrepancies between results obtained using RNA interference (RNAi)
a technique that reduces gene expression - and those from
genetic mutants. 4
Both methods have
their limitations, notes Lawson.
RNAi occasionally alters the
expression of genes
other than its desired target. And meddling with the cell's
internal RNA-processing machinery can sometimes affect other
cellular systems that involve RNA.
editing, meanwhile, requires breaking strands of DNA - which can
trigger other responses in the cell, including cell suicide.
technique can also sometimes
cut DNA at unintended sites.
Back to basics
from RNAi and genetic screens do not always mean that one approach
was right and the other was wrong, cautions Michael Bassik.
Some cells might
respond differently to a genetic change that wipes out expression of
a gene, as is often the goal with CRISPR-Cas9, compared to how they
respond to reducing the expression to very low levels with RNAi.
But often, he adds,
the culprit behind the discrepancy can be tracked back to
RNAi's potential for off-target effects. And concerns about that
have had researchers flocking to reproduce old results.
In the case of
MELK, the CRISPR-Cas9 results are particularly concerning
because they could undermine the scientific foundation for a
But Sheltzer's team showed only that MELK does not
seem to have a role in cancer-cell division, notes Carlos Moreno, a
cancer researcher at Emory University in Atlanta, Georgia.
possible that other aspects of MELK, such as its purported role in
making cancer cells more resistant to radiation, 5 are still valid,
And many successful
drugs have been developed on the back of a faulty scientific
hypothesis, he adds.
The MELK inhibitors in clinical trials might
work through some other mechanism, for instance.
"That would be
no reason to stop a trial if the trial is showing positive
effects," he says. "It's important not to throw the baby out
with the bathwater."
6, e24179 (2017)
invalidates a putative cancer dependency targeted in
on-going clinical trials,
et al. Dev. Cell
Reverse genetic screening reveals
poor correlation between morpholino-induced and mutant
phenotypes in zebrafish
et al. Proc. Natl Acad.
Sci. USA 112,
Auxin binding protein 1 (ABP1) is
not required for either auxin signaling or Arabidopsis
Systematic comparison of
CRISPR/Cas9 and RNAi screens for essential genes,
et al. Stem Cell Reports
EZH2 protects glioma stem cells
from radiation-induced cell death in a