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ơŪһʿNgAgo༭Ӿ

ӢȻ201688
ߣDavid Cyranoski

ӷ룩

һ־ݳеCRISPRCCas9ϵͳĻ༭ʵǷЧ


ǰʯׯӱƼѧһѧߺ걨˵NgAgoø
ܹڱ༭鶯ĻڣԽԽĿѧڱԹ˵޷ظ
ĽȻһߡȻ˵ظ

˵ÿյʮɧŵ绰ͶţЦְҵĽ
ˡ밴˵޷֤ʵҼɡΪвЩӳظ
ˣϴ̫ȥɧŵ绰
ǿɿġߡȻ88ӦŴϢ浵վ
AddgeneҪύһϸʵ鲽裬ϣ⽫ظ乤
밴ύʵ鲽ӵݾǰ̳Ӧ
ġҪעԤ֧ԭȾ֮࣬ʵݡоġȻ
＼ڵ¡

漰ش档ڹȥļڣCRISPRCCas9ϵͳѸıѧ
Ҳÿѧǿ༭ߣNgAgoǼѳֵ
·еһ֡ǺܶΪ件ϣСҽѧԺ
ŴѧGeorge Church˵

CRISPRCCas9һСŴһøضλиDNA
ѰܱĵʡһArgonaute
ĵʼҪ˴ǰѾ൰Ϊпڻ༭
Ǳ

ƪУŶӱ˵ʹһϵб仯Ŵϵ
Щеһ֣NgAgoܱ༭ϸеİ˸ͬ򣬲ѻ
Ⱦɫضλ㡣

˵ؼǣNgAgoԺǿֻиĿ򣬶CRISPRCCas9ʱ
༭Ļ򡣶ңCRISPRCCas9Ҫڿиλĵطһض
ŴϵиNgAgoҪܵ;
˵

йоӦһƬ̨Ҳȥʵ
Ҳɷá˵ܲһƧˡİðռҶ
١ϲУû뿪й3ȥݰݷһ
42һɻķ֮ǰû֪ҡ˵
ʵҺ͸һҲ͹ݽˡȻĲɷá

7³ԺоĻɿʼ֡ʱһԽ¶ٿѧҳ
ǰﻯѧѧ߷񣨷ӣվ˿׫˵ӵͼ
ظоŬʧܵı棬ָ˵޷ظġ
վԽԽĶԺ

729գһ˹ʡʱĴǹѧһŴѧ
Gaetan Burgio䲩ͼظʵ鵫ʧܵϸ
ֻͨмʮƪĵĵǵ˳5000

ͬһ죬＼ĵŴѧLlus Montoliu
תѧͬ·ȥʼҡκ漰ʹNgAgo
Ŀ˷ʱ䡢ǮԱʼй©ڷ
վϡ

ԺӢMRCҽѧĵķѧPooran Dewari
ϵ鷢ֻ֣9о˵NgAgoЧ밴֪9˴󲿷ˮ
5ʵͬһˣʮͶƱ97˵Ч

һȺоһʼ˵NgAgoЧ˵
ˡµCSIR-ѧѧоѧDebojyoti 
Chakraborty˵ظ˺еһֹⲿʹNgAgoóһ
תϸеıӫ⵰׵ĻӫˣChakrabortyٶNgAgo
ӫʧЧˡڶDNA˲֮ûзֻ༭֤ݡ
˵ӫļһɱصµġ

±¹֢оĻѧʿJan Winter˵Ƶľ
ҽڽļʵ飬ΪЧ˵

˵ֻʵϸиϵͳЧϸ
Чϸһֽ֧ԭϸȾ˵˿
ͬ⡣˵ЩоҲʵ̫죬ʹԼСġWinter
ͬ⣺ΪⲻǳЩظĿѧʲô

йһںоСоڲж
ԸߡȻ˵ڼϸвNgAgoҪ
λͻ䣬ò֤ʵһ˵̵Ч
ʲCRISPRCCas9ҪŻЧʡǣ򵥵˵Ч
˵밴òƾпԺϺоԱǰһֱϹ
ͦ꣬ɺˣΪʲôˡȻҪأ

йѧҲҪ˵ǳĽʾNgAgoЧ
Ҫòȷϡ밴ûвȷϣͲ˵Ч

ҲЧBurgio˵ǼˣҲ̫Ѹˣֵȥ
㡣ᳬԽCRISPRϣ̫ãˡ

NgAgoʧܡʧǻЩҪArgonauteϵͳǲ
ĳЩЧߺѧ΢ѧJohn Van der Oost˵
2014һƪArgonaute׵ĵĹͬߣƪΪڻ
༭е;춨˻

ܣȻ＼ȻŶӷһ˵
оߡϵÿ˵޷ظĽռȶ
⡱ÿһ˾ܾ۵ʻޡ

йý屨ӱƼѧ˵Ҫһظʵ鲢
ȷϡ

http://www.nature.com/news/replications-ridicule-and-a-recluse-the-controversy-over-ngago-gene-editing-intensifies-1.20387

NATURE | NEWS

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 CRISPRCCas9 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 Hans 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 Addgenes 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 CRISPRCCas9 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.

CRISPRCCas9 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.

In the paper, Hans 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, 768C773; 2016).

Crucially, NgAgo very specifically cut only the target genes, says Han, 
unlike CRISPRCCas9, which sometimes edits the wrong genes. And 
whereas CRISPRCCas9 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 
doesnt 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 Hans paper was irreproducible. Criticism grew on 
various Chinese sites.

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 Llus 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 Fangs 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 doesnt.

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.

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 Hans research 
group and who doesnt 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 CRISPRCCas9, 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, its so 
challenging that its not worth pursuing. It wont 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, 258C261; 2014).

This week, Nature Biotechnology sent a statement to Natures 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 Natures 
publisher, Springer Nature; Natures news and comment team is 
editorially independent of the publishers 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.

Nature 536, 136C137 (11 August 2016) doi:10.1038/536136a

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               ˹ܻ

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2016.3.14.

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FMĿӽѧ77ӱƴӦô麫ꣿ

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52գӢȻ־ӿȻ＼־߷˺
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ӣ߲ðɱҪߡɵĿл
ٶҡܽ¶ôҾ

Ϻרο粨ԺʿҪɱͰɱ

 2016-08-02

һվ۵ķ˼ϡλӱƼѧĸڣԭ
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