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生物科技, "CRISPR" : 一切從一個例行講座開始 (2016.10)

張鋒|Feng Zhang -  這位34歲的biomedical engineer首次在2011年的一講座中,得悉某些細菌可以翦除病毒|Viral DNA的一部分。之後他就研究如何cut and paste DNA來改變基因組|Genome, 但都一路徒勞無功,直致到他意識到細菌的作用。

於是他快速地自學了有關 CRISPR 的運作,並由“編輯”哺乳動物細胞轉移到細菌類細胞, 風險是在細菌中工作的系統,在遺傳學上比哺乳動物簡單得很多。但在兩年內,他發表了關於第一次使用CRISPR從小鼠和人類細胞的前所未有的控制特定部分的DNA中剔除的里程碑報告。他的技術啟動了一項 CRISPR (註1) 的革命,例如允許科學家將感染愛艾滋病毒的細胞或受基因突變致病的細胞中的部份基因分離出來,這改變一切了! 他說,“突然之間,科學有能力對DNA進行明確的改變 - 對醫學,基礎生物學到植物學等發展急速地成為一個催化劑。”一切都從一個普通講座開始。

張鋒博士的最新突破是什麼?

簡單來說,研究人員們發現了一個新興CRISPR系統。事實上,除了CRISPR/Cas系統,CRISPR系統的類型眾多。 Cas9僅僅是其中一類作用蛋白,CRISPR序列理論上還可以跟許多其它蛋白共同作用。但是長期以來,這些系統並未被驗證可以進行有效的基因編輯。 CRISPR/C2c2 就是張鋒博士的團隊最新發現的可以被用作基因編輯的CRISPR系統。

這個系統的突破性意義就在於它可以在RNA級別進行編輯,而不是傳統的DNA級別的編輯。該系統基本的工作流程與CRISPR/Cas9類似,還是藉助CRISPR序列的”黑名單“系統對入侵者進行打擊。但是crRNA形成的方式與CRISPR/Cas9系統不同。 C2c2蛋白會與成熟的crRNA複合,在不借助tracrRNA的情況下與外源單鏈RNA結合,crRNA則會與PFS片段(類似PAM)附近的互補區域雜交。最後,外源單鏈RNA會被剪切,其基因表達也會被滅掉。

然而,被剪切的外源RNA有可能會觸發宿主細胞RNA的傷害,從而引發宿主細胞凋亡機制。這種只靶向作用於RNA並協助執行基因組指令的能力能夠讓人們特異性地和高通量地操縱RNA,以及更加廣泛地操縱基因功能。這技術有潛力加快認適、治療和預防疾病的步伐。張博士說,“C2c2為CRISPR工具打開另一個大門。對C2c2而言,它有大量的可能性,而且我們興奮地將它開發為一種用於生命科學研究和醫學的平台。 ”

有興趣的,這頁的英文版有更精彩的內容! 請按此或右上角的 [ English Version ]

註1: 有關 CRISPR

CRISPR全稱 ”Clustered Regularly Interspersed Short Palindromic Repeats |有規律性集群的穿插中的短回文重複序列”,分佈在40%的已測序細菌和90%的已測序古細菌當中。CRISPR是存在於細菌和古生菌基因組中的特殊DNA重複序列家族。

CRISPR/Cas系統是一種原核生物的免疫系統,用來抵抗外源遺傳物質的入侵,比如噬菌體病毒和外源質粒。同時,它為細菌提供了獲得性免疫:這與哺乳動物的二次免疫類似,當細菌遭受病毒或者外源質粒入侵時,會產生相應的“記憶”,從而可以抵抗它們的再次入侵。 CRISPR/Cas系統可以識別出外源DNA,並將它們切斷,沉默外源基因的表達。這與真核生物中RNA干擾(RNAi)的原理是相似的。

由於這種精確的靶向功能,CRISPR/Cas系統被開發成一種高效的基因編輯工具。在自然界中,CRISPR/Cas系統擁有多種類別,其中CRISPR/Cas9系統是研究最深入,應用最成熟的一種類別。憑高效率,CRISPR/Cas9迅速風靡全球的實驗室,人們就可以使用CRISPR/Cas9技術輕鬆的實現基因編輯。

註2:  有關 CRISPR的發展史 (抱謙, 祗提供英文)

CRISPR/Cas9 is a genetic engineering technique that makes use of the cellular machinery that bacteria use to recognise and edit the DNA of harmful viruses. It has two key components: Cas9, an enzyme that can cut a double DNA strand at a very precise point, nick it, or block its gene expression; and CRISPR, a short strand of RNA, a chemical messenger. The CRISPR helps guide the Cas9 enzyme to bind with a specific sequence on the genome where it will make a specific cut. By this means scientists can either splice out part of a gene to disrupt its function, or insert a new sequence into the genome to code for a new function.

 

Date

Event

People

Places

December 1987

Discovery of CRISPR mechanism

Amemura, Ishino, Makino, Nakata, Shinagawa, Takase, Wachi

Osaka University

January 18, 2000

More clustered repeats of DNA identified in other bacteria and archaea, termed Short Regularly Spaced Repeats (SRSR)

Mojica, Diez-Villasenor, Soria, Juez

University of Alicante, University Miguel Hernandez

March 2002

Dutch scientists coin the term CRISPR-Cas9. Strands for clustered regularly interspaced short palindrome repeats in a DNA sequence and its associated genes.

Jansen, Embden, Gaastra, Schouls

Utrecht University

August 2005

French scientists suggest CRISPR spacer sequences provide cell immunity against phage infection and are a means of degrading DNA

Bolotin, Quinquis, Sorokin, Ehrlich

Institut National de la Recherche Agronomique

November 2005

American researchers identified new familes of Cas genes which appeared to help in protecting bacteria against invading viruses

Haft, Selengut, Mongodin, Nelson

The Institute for Genomic Research

March 2007

Experiments conducted by microbiologists based at the food manufacturing company Danisco, demonstrate for the first time the role of CRISPR together with Cas9 genes in protecting bacteria against viruses

Barrangou, Horvath, Fremaux, Deveau,

Danisco USA Inc

2008

DNA, not RNA, demonstrated to be the molecular target of most CRISPR-Cas systems

   

February 2008

Scientists coin the term 'protospacer' to denote viral sequence that corresponds to a 'spacer' in the CRISPR-Cas9 system

   

August 2008

Scientists characterised the RNA processing pathway in CRISPR system

 

Wageningen University, University of Sheffield, National Institutes of Health

December 2008

Scientists work out the RNA gene silencing pathway involved in the CRISPR-Cas mechanism

 

University of Georgia, Florida State University

2011

Classification of the CRISPR-Cas system is proposed

   

April 2012

DuPont commercialises the first bacterial cultures based on the CRISPR-Cas 9 technology for the production of pizza cheese

 

Dupont

May 2012

First patent application submitted for CRISPR-Cas 9 technology

Doudna, Charpentier

University of California Berkeley, University of Vienna

August 2012

A group of scientists based at Howard Hughes Medical Institute publish a new method that harnesses the CRISPR-Cas9 system for genome editing

Jinek, Chylinski, Fonfara, Hauer, Doudna, Charpentier

University of California Berkeley

December 2012

Fast track application for CRISPR-Cas 9 technology submitted to US patent office.

Zhang

Broad Institute, Massachusetts Institute of Technology

January 2013

CRISPR-Cas is used in human genome editing

   

January 2013

CRISPR-Cas is used to edit the genome of a zebrafish

   

February 2013

CRISPR-Cas is used to programme repression and activation of gene transcription

   

February 2013

Scientists from Harvard Medical School announce the successful genetic engineering of human cells using a CRISPR-Cas 9 technique

Mali, Yang, Esvelt, Aach, Guell, DiCarlo, Norville, Church

Harvard University

March 2013

CRISPR-Cas is used in genome editing of Saccharomyces cerevisiae, a yeast species used in wine making, baking and brewing

   

April 2013

Scientists show that CRISPR-Cas mediated gene regulation contributes to the regulation of endogenous bacterial genes

Weiss

 

August 2013

CRISPR-Cas used to engineer a rat's genome

   

August 2013

CRISPR-Cas used to engineer plant genomes including rice, wheat, Arabidopsis, tobacco and Sorghum

   

August 2013

Improvements made to the specificity of CRISPR-Cas system

   

March 2014

First patent awarded for CRISPR-Cas9 technology to be used for a wide range of applications

Zhang

Broad Institute, Massachusetts Institute of Technology

March 2015

Scientists suggest the use of CRISPR/Cas9 in conjunction with stem cells to provide human organs from transgenic pigs

Feng, Dai, Mou, Cooper, Shi, Cai

Shenzhen University, University of Pittsburgh Medical Center, Guangxi University

March 26, 2015

US scientists call for a voluntary worldwide moratorium on the use of genome editing tools to modify human reproductive cells

Lamphier, Urnov

 

April 2015

National Institutes of Health declares it will not fund any use of genome editing technologies in human embryos

   

April 22, 2015

UK Nuffield Council on Bioethics launched a new working group to look into institutional, national and international policies and provisions relevant to genome editing

   

May 2015

Chinese scientists, led by Junjiu Huang, publish first experiments modifying the genome of human embryos with CRISPR-Cas9 technique, prompting bioethical debate over use of technology

Huang, Liang, Xu, Zhang

Sun Yat-sen University

September 2015

UK Medical Research Council and other leading UK research councils declared support for using CRISPR-Cas9 and other genome editing techniques in preclinical research

   

September 11, 2015

Hinxton Group issues a statement indicating that most of the ethical and moral questions raised about genome editing so have have been debated before.

   

September 15, 2015

UK Nuffield Council on Bioethics held its first workshop to identify and define ethical questions relating to developments in genome editing research

   

September 18, 2015

UK scientists seek license to genetically modify human embryos to study the role played by genes in the first few days of human fertilisation

Naikan

Crick Institute

September 25, 2015

New protein, Cpf1, found which could simplify genome editing.

Zhang, Zetsche, Gootenberg, Abudayyeh, Slaymaker

Broad Institute, Massachusetts Institute of Technology

October 5, 2015

US Scientists report use of CRISPR to modify 60 genes in pig embryos in first step to create pig organs suitable for use in humans

Church

Harvard University

October 6, 2015

UNESCO’s International Bioethic Committee called for ban on genetic editing of human germline

   

November 16, 2015

US Scientists publish a technique for overwriting changes made by CRISPR/Cas 9

DiCarlo, Chavez, Dietz, Esvelt, Church

Harvard University, Swiss Federal Institute of Technology in Zurich

November 23, 2015

US scientists report successful use of CRISPR/Cas9 to genetically modify mosquitoes to make them poor hosts for malaria parasite

Gantz, Jasinskiene, Tatarenkova, Fazekas, Macias, Bier, James

University California San Diego, University of California Irvine

December 1, 2015 - December 3, 2015

International Summit on Human Gene Editing met to discuss the scientific, medical, ethical, and governance issues associated with recent advances in human gene-editing research

Baltimore, Doudna, Church, Zhang

US National Academies of Science, Engineering and Medicine, US National Academy of Medicine, Chinese Academy of Sciences, Royal Society

December 31, 2015

CRISPR successfully used to improve muscle function in mouse model of Duchenne muscular dystrophy, opening way to use CRISPR to correct genetic mutatiuons in affected tissues of sick patients

Nelson, Gersbach, Hakim, Ousterout, Thakore

Duke University, University of Missouri, University of North Carolina, Massachusetts Institute of Technology, Harvard University

January 6, 2016

Harvard scientists publish improved version of CRISPR/Cas 9 with less risk of off-target DNA breaks

Kleinstiver, Pattanayak, Prew, Tsai, Nguyen, Zheng, Joung

Harvard University

February 1, 2016

UK scientists authorised to genetically modify human embryos using CRISPR-Cas 9

Niakan

Crick Institute

       

 

參考資料 Source of information

基因組的詞彙表 (視頻) A Talking Glossary of Genome

https://www.genome.gov/glossary/

有關張鋒Regarding Feng Zhang

https://zh.wikipedia.org/wiki/%E5%BC%B5%E9%8B%92

https://en.wikipedia.org/wiki/Feng_Zhang

http://time.com/4518815/feng-zhang-next-generation-leaders/

http://time.com/4379503/crispr-scientists-edit-dna/?iid=sr-link3 )

什麼是生物科技What is Biotechnology?

http://www.whatisbiotechnology.org/science/crispr

https://www.technologyreview.com/lists/innovators-under-35/2013/pioneer/feng-zhang/

什麼是CRISPR , 為什麼這麼熱?What is CRISPR / Cas9 and why everyone is suddenly on ? 

http://alwareness.com/en/wat-is-crisprcas9-en-waarom-heeft-iedereen-het-er-opeens-over/

編輯基因的年代The Age of Gene Editing: Everything You Need To Know About CRISPR/Cas9

http://futurism.com/the-age-of-gene-editing-everything-you-need-to-know-about-crisprcas9/

CRISPR-Cas9在癌症生物學的應用Applications of the CRISPR–Cas9 system in cancer biology

http://www.nature.com/nrc/journal/v15/n7/full/nrc3950.html

CRISPR系統在原核免疫中的作用CRISPR systems in prokaryotic immunity

http://rna.berkeley.edu/crispr.html

https://en.wikipedia.org/wiki/CRISPR

其它Others (Descriptions with diagrams etc.)

http://www.london-science.com/archives/220

http://www.ebiotrade.com/newsf/2016-6/201661155130939.htm

http://www.bioon.com/trends/news/615569.shtml

 

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