Nature News: The controversy over NgAgo# Biology - 生物学
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http://www.nature.com/news/replications-ridicule-and-a-recluse-the-controversy-over-ngago-gene-editing-intensifies-1.20387
Replications, ridicule and a recluse: the controversy over NgAgo gene-
editing intensifies
As failures to replicate results using the CRISPR alternative stack up, a
quiet scientist stands by his claims.
David Cyranoski
08 August 2016
SHIJIAZHUANG, CHINA
A controversy is escalating over whether a gene-editing technique proposed
as an alternative to the popular CRISPR–Cas9 system actually works.
Three months ago, Han Chunyu, a biologist at Hebei University of Science and
Technology in Shijiazhuang, reported that the enzyme NgAgo can be used to
edit mammalian genes. Now, an increasing number of scientists are
complaining that they cannot replicate Han’s results — although one has
told Nature that he can.
Han says he receives dozens of harassing calls and texts each day, mocking
him and telling him that his career is over — but he is convinced that the
technique is sound. He also told Nature that he had submitted a detailed
protocol to the online genetic-information repository Addgene on 8 August,
at Addgene’s request, and hopes that this will help efforts to reproduce
his work. Nature Biotechnology, which published the research, is
investigating the matter.
The stakes are high. Over the past few years, the CRISPR–Cas9 system has
transformed biology. But it has also made scientists hungry for other
methods to expand the gene-editing toolkit: NgAgo is one of several that
have emerged. “A lot of us are really cheerleading and hoping that it works
,” says geneticist George Church of Harvard Medical School in Boston,
Massachusetts.
CRISPR–Cas9 uses small genetic sequences to guide an enzyme to cut DNA in a
particular location. Inspired, Han looked through the literature for other
guidable protein ‘scissors’, and came across a family of proteins called
Argonaute, or Ago, that fitted the bill. Others had flagged the proteins as
potential gene editors.
Former biochemist Fang Shimin alleged on his website that the NgAgo paper
was irreproducible.
In the paper, Han’s team reports using a wide variety of genetic sequences
to guide one of these proteins, NgAgo, to edit eight different genes in
human cells and to insert genes at specific points on chromosomes (F. Gao et
al. Nature Biotechnol. 34, 768–773; 2016).
Crucially, NgAgo very specifically cut only the target genes, says Han,
unlike CRISPR–Cas9, which sometimes edits the wrong genes. And whereas
CRISPR–Cas9 requires a certain genetic sequence to be near the cutting site
to initiate its activity, NgAgo does not, which could broaden its potential
applications, adds Han.
The initial reaction to the work in China was laudatory, including a visit
to the lab by China Central Television. It was overwhelming, says Han, who
is a reclusive figure. His hobbies include collecting teas and playing an
ancient stringed instrument called the guqin. He doesn’t like to travel and
has never left China: a trip to visit a collaborator in Hangzhou in March
was the first time the 42 year old had boarded a plane. Before his paper
came out, “I was completely unknown”, says Han, who spoke to Nature at his
laboratory and a nearby restaurant.
Doubts about the research first surfaced at the beginning of July, when Fang
Shimin, a former biochemist who has become famous for exposing fraudulent
scientists, wrote on his website New Threads (xys.org) that he had heard
reports of failed reproduction efforts, and alleged that Han’s paper was
irreproducible. Criticism grew on various Chinese sites.
Geneticist Gaetan Burgio published failed attempts to replicate the NgAgo
experiments on his blog.
On 29 July, the controversy went inter-national when Gaetan Burgio, a
geneticist at the Australian National University in Canberra, posted
thorough details of his failed attempts to replicate the experiment on his
blog. Normally, his posts get a few dozen hits, but this one spiked to more
than 5,000.
On the same day, geneticist Lluís Montoliu, at the Spanish National Centre
for Biotechnology in Madrid, e-mailed his colleagues at the International
Society for Transgenic Technologies to recommend “abandoning any project
involving the use of NgAgo” to “avoid wasting time, money, animals and
people”. The e-mail was leaked and posted on Fang’s website.
Since then, an online survey by Pooran Dewari, a molecular biologist at the
MRC Centre for Regenerative Medicine in Edinburgh, UK, has found only 9
researchers who say that NgAgo works — and 97 who say that it doesn’t.
Two researchers who initially reported success with NgAgo in an online chat
group now say that they were mistaken. Debojyoti Chakraborty, a molecular
biologist at the CSIR-Institute of Genomics and Integrative Biology in New
Delhi, says that he repeated a specific section of Han’s paper that
described using NgAgo to knock out a gene for a fluorescent protein that had
been introduced into a cell. The glow was reduced, so Chakraborty assumed
that NgAgo had disabled the gene. But after sequencing the DNA, he found no
evidence of gene editing. He now says that the reduction in fluorescence
must have had some other cause.
Jan Winter, a PhD student in genomics at the German Cancer Research Center
in Heidelberg, says that he had a similar experience. “I will retry the
experiment in the upcoming weeks, but so far I think it won’t work,” he
says.
Geneticist Lluís Montoliu recommends abandoning any project involving the
use of NgAgo for gene-editing.
Han says that he has only got the system to work on cells cultured in his
laboratory, and it failed in cells that he purchased. He later found the
purchased cells to be contaminated with bacteria called mycoplasma, and says
that others might be having the same problem. He adds that some graduate
students might be working too fast and not being careful with reagents.
Winter disagrees: “I do not think it is a problem of the scientists doing
something wrong.”
One researcher in China, who works independently from Han’s research group
and who doesn’t want his name to be entangled in the public controversy,
told Nature that he had tested NgAgo in a few kinds of cell and found that
it was able to induce genetic mutations at the desired sites — a finding
that he verified by sequencing. He adds that the process was less efficient
than CRISPR–Cas9, and requires tweaking to improve the efficiency. “But,
in short, it worked,” he says.
Two more Chinese scientists, who also asked not to be named, say they have
initial results showing that NgAgo works but still need to confirm with
sequencing.
“It might, might work,” says Burgio, “but if so, it’s so challenging
that it’s not worth pursuing. It won’t surpass CRISPR, not by a long shot.”
The failure of NgAgo “would be disappointing, but then there is work for us
left to do to see whether other Argonaute systems can get it to work
somehow,” says microbiologist John Van der Oost of Wageningen University in
the Netherlands, a co-author of the 2014 analysis of Argonaute proteins
that laid the groundwork for their use in gene editing (D. C. Swaarts et al.
Nature 507, 258–261; 2014).
This week, Nature Biotechnology sent a statement to Nature’s news team,
saying that “several researchers” have contacted the journal to report
that they cannot reproduce the results, and that “the journal is following
established process to investigate the issues”. A spokesperson declined to
comment on the nature or duration of the investigation. (Nature
Biotechnology is published by Nature’s publisher, Springer Nature; Nature’
s news and comment team is editorially independent of the publisher’s
research editorial teams.)
Hebei University says that it will ask Han to repeat the experiment so that
it can be verified by an independent party within a month, according to
Chinese state media.
Replications, ridicule and a recluse: the controversy over NgAgo gene-
editing intensifies
As failures to replicate results using the CRISPR alternative stack up, a
quiet scientist stands by his claims.
David Cyranoski
08 August 2016
SHIJIAZHUANG, CHINA
A controversy is escalating over whether a gene-editing technique proposed
as an alternative to the popular CRISPR–Cas9 system actually works.
Three months ago, Han Chunyu, a biologist at Hebei University of Science and
Technology in Shijiazhuang, reported that the enzyme NgAgo can be used to
edit mammalian genes. Now, an increasing number of scientists are
complaining that they cannot replicate Han’s results — although one has
told Nature that he can.
Han says he receives dozens of harassing calls and texts each day, mocking
him and telling him that his career is over — but he is convinced that the
technique is sound. He also told Nature that he had submitted a detailed
protocol to the online genetic-information repository Addgene on 8 August,
at Addgene’s request, and hopes that this will help efforts to reproduce
his work. Nature Biotechnology, which published the research, is
investigating the matter.
The stakes are high. Over the past few years, the CRISPR–Cas9 system has
transformed biology. But it has also made scientists hungry for other
methods to expand the gene-editing toolkit: NgAgo is one of several that
have emerged. “A lot of us are really cheerleading and hoping that it works
,” says geneticist George Church of Harvard Medical School in Boston,
Massachusetts.
CRISPR–Cas9 uses small genetic sequences to guide an enzyme to cut DNA in a
particular location. Inspired, Han looked through the literature for other
guidable protein ‘scissors’, and came across a family of proteins called
Argonaute, or Ago, that fitted the bill. Others had flagged the proteins as
potential gene editors.
Former biochemist Fang Shimin alleged on his website that the NgAgo paper
was irreproducible.
In the paper, Han’s team reports using a wide variety of genetic sequences
to guide one of these proteins, NgAgo, to edit eight different genes in
human cells and to insert genes at specific points on chromosomes (F. Gao et
al. Nature Biotechnol. 34, 768–773; 2016).
Crucially, NgAgo very specifically cut only the target genes, says Han,
unlike CRISPR–Cas9, which sometimes edits the wrong genes. And whereas
CRISPR–Cas9 requires a certain genetic sequence to be near the cutting site
to initiate its activity, NgAgo does not, which could broaden its potential
applications, adds Han.
The initial reaction to the work in China was laudatory, including a visit
to the lab by China Central Television. It was overwhelming, says Han, who
is a reclusive figure. His hobbies include collecting teas and playing an
ancient stringed instrument called the guqin. He doesn’t like to travel and
has never left China: a trip to visit a collaborator in Hangzhou in March
was the first time the 42 year old had boarded a plane. Before his paper
came out, “I was completely unknown”, says Han, who spoke to Nature at his
laboratory and a nearby restaurant.
Doubts about the research first surfaced at the beginning of July, when Fang
Shimin, a former biochemist who has become famous for exposing fraudulent
scientists, wrote on his website New Threads (xys.org) that he had heard
reports of failed reproduction efforts, and alleged that Han’s paper was
irreproducible. Criticism grew on various Chinese sites.
Geneticist Gaetan Burgio published failed attempts to replicate the NgAgo
experiments on his blog.
On 29 July, the controversy went inter-national when Gaetan Burgio, a
geneticist at the Australian National University in Canberra, posted
thorough details of his failed attempts to replicate the experiment on his
blog. Normally, his posts get a few dozen hits, but this one spiked to more
than 5,000.
On the same day, geneticist Lluís Montoliu, at the Spanish National Centre
for Biotechnology in Madrid, e-mailed his colleagues at the International
Society for Transgenic Technologies to recommend “abandoning any project
involving the use of NgAgo” to “avoid wasting time, money, animals and
people”. The e-mail was leaked and posted on Fang’s website.
Since then, an online survey by Pooran Dewari, a molecular biologist at the
MRC Centre for Regenerative Medicine in Edinburgh, UK, has found only 9
researchers who say that NgAgo works — and 97 who say that it doesn’t.
Two researchers who initially reported success with NgAgo in an online chat
group now say that they were mistaken. Debojyoti Chakraborty, a molecular
biologist at the CSIR-Institute of Genomics and Integrative Biology in New
Delhi, says that he repeated a specific section of Han’s paper that
described using NgAgo to knock out a gene for a fluorescent protein that had
been introduced into a cell. The glow was reduced, so Chakraborty assumed
that NgAgo had disabled the gene. But after sequencing the DNA, he found no
evidence of gene editing. He now says that the reduction in fluorescence
must have had some other cause.
Jan Winter, a PhD student in genomics at the German Cancer Research Center
in Heidelberg, says that he had a similar experience. “I will retry the
experiment in the upcoming weeks, but so far I think it won’t work,” he
says.
Geneticist Lluís Montoliu recommends abandoning any project involving the
use of NgAgo for gene-editing.
Han says that he has only got the system to work on cells cultured in his
laboratory, and it failed in cells that he purchased. He later found the
purchased cells to be contaminated with bacteria called mycoplasma, and says
that others might be having the same problem. He adds that some graduate
students might be working too fast and not being careful with reagents.
Winter disagrees: “I do not think it is a problem of the scientists doing
something wrong.”
One researcher in China, who works independently from Han’s research group
and who doesn’t want his name to be entangled in the public controversy,
told Nature that he had tested NgAgo in a few kinds of cell and found that
it was able to induce genetic mutations at the desired sites — a finding
that he verified by sequencing. He adds that the process was less efficient
than CRISPR–Cas9, and requires tweaking to improve the efficiency. “But,
in short, it worked,” he says.
Two more Chinese scientists, who also asked not to be named, say they have
initial results showing that NgAgo works but still need to confirm with
sequencing.
“It might, might work,” says Burgio, “but if so, it’s so challenging
that it’s not worth pursuing. It won’t surpass CRISPR, not by a long shot.”
The failure of NgAgo “would be disappointing, but then there is work for us
left to do to see whether other Argonaute systems can get it to work
somehow,” says microbiologist John Van der Oost of Wageningen University in
the Netherlands, a co-author of the 2014 analysis of Argonaute proteins
that laid the groundwork for their use in gene editing (D. C. Swaarts et al.
Nature 507, 258–261; 2014).
This week, Nature Biotechnology sent a statement to Nature’s news team,
saying that “several researchers” have contacted the journal to report
that they cannot reproduce the results, and that “the journal is following
established process to investigate the issues”. A spokesperson declined to
comment on the nature or duration of the investigation. (Nature
Biotechnology is published by Nature’s publisher, Springer Nature; Nature’
s news and comment team is editorially independent of the publisher’s
research editorial teams.)
Hebei University says that it will ask Han to repeat the experiment so that
it can be verified by an independent party within a month, according to
Chinese state media.
