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CRISPR-Cas9
De nition
CRISPR/Cas9 is a technique that allows for the highly specific and rapid modification of DNA in a genome, the complete set
of genetic instructions in an organism.
This image depicts genome editing. It is adapted from a DNA illustration by Spooky Pooka. Credit: Wellcome Images.
Connections Jennifer Doudna | Recombinant DNA | Transgenic animals

Importance
The CRISPR/Cas 9 technique is one of a number of gene-editing tools. Many favour the CRISPR/Cas9 technique because of
its high degree of flexibility and accuracy in cutting and pasting DNA. One of the reasons for its popularity is that it makes
it possible to carry out genetic engineering on an unprecedented scale at a very low cost. How it differs from previous
genetic engineering techniques is that it allows for the introduction or removal of more than one gene at a time. This
makes it possible to manipulate many different genes in a cell line, plant or animal very quickly, reducing the process from
taking a number of years to a matter of weeks. It is also different in that it is not species-specific, so can be used on
organisms previously resistant to genetic engineering.

The technique is already being explored for a wide number of applications in fields ranging from agriculture through to
human health. In agriculture it could help in the design of new grains, roots and fruits. Within the context of health it could
pave the way to the development of new treatments for rare metabolic disorders and genetic diseases ranging from
haemophilia through to Huntingdon's disease. It is also being utilised in the creation of transgenic animals to produce
organs for transplants into human patients. The technology is also being investigated for gene therapy. Such therapy aims
to insert normal genes into the cells of people who suffer from genetic disorders such as cystic fibrosis, haemophilia or Tay
Sachs. Several start-up companies have been founded to exploit the technology commercially and large pharmaceutical
companies are also exploring its use for drug discovery and development purposes.

Discovery
In 1987 a Japanese team of scientists at Osaka University noticed a strange pattern of DNA sequences in a gene belonging
to Escherichia coli, a microbe that lives in the gut. It appeared that the gene had five short repeating segments of DNA
separated by short non-repeating 'spacer' DNA sequences. All five repeating segments had identical sequences composed
of 29 bases, the building blocks of DNA. By contrast each of the 'spacer' sequences had their own unique sequence,
composed of 32 bases. Microbiologists had never seen such a pattern before. By the end of the 1990s, however, they had
begun to discover, with the aid of new improvements to DNA sequencing, that this pattern was prevalent in many different
microbe species.

So common was the pattern that it was given its own name: 'clustered regularly inter-spaced short palindromic repeats' or
CRISPR for short. The term was coined by a team of Dutch scientists led by Rudd Jansen at Utrecht University, in 2002,
who the same year noted that another set of sequences always accompanied the CRISPR sequence. This second set of
sequences they dubbed 'Cas genes', an abbreviation for CRISPR-associated genes. The Cas genes appeared to code for
enzymes that cut DNA. By 2005 three scientific teams had independently worked out that the 'spacer' sequences between
the CRISP sequences shared similarities with the DNA of viruses and hypothesised that it could be a tool in the defence
mechanism of bacteria.

Knowledge about how the CRISPR/Cas 9 system worked was opened up by some experiments conducted in 2007 by
scientists at Danisco, a Danish food manufacturer later acquired by DuPont. The team infected a milk-fermenting microbe,
Streptococcus thermophilius, with two virus strains. Many of these bacteria were killed by the viruses, but some survived
and went on to produce offspring also resistant to the viruses. On further investigation it appeared that the microbes were
inserting DNA fragments from the viruses into their 'spacer' sequences and that they lost resistance whenever the new
'spacer' sequences were cut out.

In 2008 Eugene Koonin and colleagues at the National Center for Biotechnology Information in Bethesda, Maryland,
demonstrated for the first time how the CRISPR/Cas 9 mechanism worked. Whenever bacteria confront an invader, such as
a virus, they copy and incorporate its DNA segments into their genome as 'spacers' between the short DNA repeats in
CRISPR. The segments in the 'spacers' provide a template for the bacteria's RNA molecules to recognise any future DNA of
an incoming virus and help guide the Cas 9 enzyme to cut it up so as to disable the virus.

Four years later, in 2012, a small team of scientists led by Jennifer Doudna, University California Berkeley, and Emmanuelle
Charpentier, University of Umea, published a paper showing how to harness the natural CRISPR-Cas9 system as a tool to
cut any DNA strand in a test tube. A year later, in January 2013, a number of researchers at different laboratories published
papers within a few weeks of each other demonstrating how the CRISPR/Cas 9 system could be used to edit genomes in
human cells. This included teams led by Doudna, Feng Zhang at MIT-Harvard Broad Institute, and George Church at Harvard
Medical School.

A number of changes are now underway to improve the accuracy and efficiency of the CRISPR-Cas 9 technique. A key
breakthrough has been the development of new Cas9 fusion proteins to act as base editors. The fusion proteins make it
possible to convert cytosine to uracil without cutting DNA. Uracil is subsequently transformed into thymine through DNA
replication or repair. The first base editors were generated in 2016 by Alexis Komor and colleagues in the laboratory of
David Liu at Harvard University.
Application
The CRISPR/Cas 9 system was first exploited by Danisco in 2008. The company used it to improve the immunity of
bacterial cultures against viruses and many food manufacturers now use the technology to produce cheese and yoghurt.
Since then the technology has been used to delete, insert and modify DNA in human cells and other animal cells grown in
petri dishes. Scientists are also using it to create transgenic animals such as mice, rats, zebrafish, pigs and primates.
Between 2014 and 2015 scientists reported the successful use of CRISPR/Cas 9 in mice to eliminate muscular dystrophy
and cure a rare liver disease, and to make human cells immune to HIV. It is also being investigated in conjunction with
pluripotent stem cells to provide human organs from transgenic pigs. Such work is directed towards helping solve some of
the shortage of human organs for transplant operations and overcome some of the side-effects caused by organ
transplantation such as graft-versus host disease. The technology is also being investigated as a means to genetically
engineer insects so as to wipe out insect-borne diseases such as malaria, transmitted by mosquitoes, and lyme disease,
transmitted by ticks.

Issues
In April 2015 a Chinese group reported the first application of CRISPR/Cas9 to (non-viable) human embryos. This
development, together with the decreasing costs of the technology have triggered a major bioethical debate about how far
the technology should be used. The technology faces two major issues.

The first issue is a philosophical dilemma. It centres on the extent to which CRISPR/Cas9 should be used to alter 'germ-
line' cells - eggs and sperm - which are responsible for passing genes on to the next generation. While it will take many
more years before the technology will be viable to use to create designer babies, a public debate has already begun on this
issue. So great is the fear that some scientists, including some who helped pioneer CRISPR/Cas9, have called for a
moratorium on its use in germ-line cells.

The second issue is one of safety. One of the major problems is that the technology is still in its infancy and knowledge
about the genome remains very limited. Many scientists caution that the technology still needs a lot of work to increase
its accuracy and make sure that changes made in one part of the genome do not introduce changes elsewhere which could
have unforeseen consequences. This is a particularly important issue when it comes to the use of the technology for
applications directed towards human health. Another critical issue is that once an organism, such as a plant or insect, is
modified they are difficult to distinguish from the wild-type and once released into the environment could endanger
biodiversity.

Policy-makers are still debating about what limitations to put on the technology. In April 2015 the US National Institutes of
Health issued a statement indicating that it will not fund any research that uses genome editing tools such as CRISPR in
human embryos. Meanwhile, the UK's Human Fertilisation and Embryology Authority, under whose remit such research
would fall, has indicated that the CRISPR/Cas9 technology can be used on human-animal hybrid embryos under 14 days old.
Any researcher working in this area would need to first get a license from the Authority. Other leading UK research councils
have indicated that they support the continued use of CRISPR/Cas9 and other genome editing tools in preclinical research.

As regulators debate what restrictions to enforce with CRISPR/Cas9, the technology has become the subject of a major
patent dispute. The first application to patent the technology was filed by DuPont in March 2007 (WO/2007/025097). This
covers the use of the technology to develop phage resistant bacterial strains for food production, feeds, cosmetics,
personal care products and veterinary products. Since then three heavily financed start-up biotechnology companies and
half a dozen universities have filed patents. Two major competing patent claims have been filed in the US. The first, filed on
25 May 2015, is grounded in the work led by Jennifer Doudna at the University of California, Berkeley, and Emmanuelle
Charpentier, originally at the University of Vienna and now at the Helmholz Centre for Infectious Research in Germany. The
application has 155 claims and covers numerous applications for a variety of cell types (US Patent Application No.
PCT/US2013/032589). The second, was filed by MIT-Harvard Broad Institute on 12 December 2012 for the work of Feng
Zhang which focused on the use of CRISPR/Cas9 for genome editing in eukaryotic cells. It was given fast-track status and
was granted on 15 April 2014 (US Patent No. 8,697,359). In April 2015 Charpentier and the Universities of California and
Vienna filed a challenge to the patent with the US Patent and Trademark Office. It will take several years for the patent
dispute to be settled.

This profile was written by Lara Marks in June 2016 with generous input from Silvia Camporesi, Xiofan Zeng and Jonathan
Lind.

CRISPR-Cas9: timeline of key events

Date Event People Places
December 1987 The CRISPR mechanism first published Amemura, Ishino, Makino, Osaka University
Nakata, Shinagawa,
Takase, Wachi
18 Jan 2000 More clustered repeats of DNA identified in other Mojica, Diez-Villasenor, University of Alicante,
bacteria and archaea, termed Short Regularly Soria, Juez University Miguel
Spaced Repeats (SRSR) Hernandez
March 2002 Term CRISPR-Cas9 published for first time Mojica, Jansen, Embden, Utrecht University
Gaastra, Schouls
2005 Jennifer Doudna and Jillian Banfield started Doudna, Banfield University of California
investigating CRISPR Berkeley
1 Aug 2005 French scientists suggested CRISPR spacer Bolotin, Quinquis, Sorokin, Institut National de la
sequences can provide cell immunity against phage Ehrlich Recherche Agronomique
infection and degrade DNA
11 Nov 2005 American researchers identified new familes of Cas Haft, Selengut, Mongodin, The Institute for Genomic
genes which appeared to help in protecting bacteria Nelson Research
against invading viruses
23 Mar 2007 Experiments demonstrate for the first time the role Barrangou, Horvath, Danisco USA Inc
of CRISPR together with Cas9 genes in protecting Fremaux, Deveau,
bacteria against viruses
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 Wageningen University,
pathway in CRISPR system University of Sheffield,
National Institutes of
Health
December 2008 Scientists published the RNA gene silencing Carte, Wang, Li, Terns University of Georgia,
pathway involved in the CRISPR-Cas mechanism Florida State University
2011 Classification of the CRISPR-Cas system is  
proposed
March 2011 Emmanuelle Charpentier and Jennifer Doudna joined Doudna, Charpentier, University of California
forces to investigate Cas9 enzyme Hinek, Hauser Berkeley, Umea University
April 2012 First commercialisation of CRISPR-Cas 9 Dupont
technology
May 2012 First patent application submitted for CRISPR-Cas 9 Doudna, Charpentier University of California
technology Berkeley, University of
Vienna
17 Aug 2012 Publication of radically new gene editing method Jinek, Chylinski, Fonfara, University of California
that harnesses the CRISPR-Cas9 system Hauer, Doudna, Berkeley
Charpentier
12 Dec 2012 Fast track application for CRISPR-Cas 9 technology Zhang Broad Institute,
submitted to US patent office. 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 shown to programme repression and Bikard, Murrafini Rockefeller University
activation of gene transcription
March 2013 CRISPR-Cas is used in genome editing of  
Saccharomyces cerevisiae, a yeast species used in
wine making, baking and brewing
1 Apr 2013 CRISPR-Cas mediated gene regulation shown to Sampson, Weiss Emory University
help regulation of endogenous bacterial genes
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 2015 Scientists suggest CRISPR/Cas9 used with stem Feng, Dai, Mou, Cooper, Shenzhen University,
cells could provide human organs from transgenic Shi, Cai University of Pittsburgh
pigs Medical Center, Guangxi
University
26 Mar 2015 US scientists call for a voluntary worldwide Lamphier, Urnov  
moratorium on the use of genome editing tools to
modify human reproductive cells
15 Apr 2015 National Institutes of Health declared it will not  
fund any use of genome editing technologies in
human embryos
22 Apr 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
1 May 2015 First report of genes edited in human embryos Huang, Liang, Xu, Zhang Sun Yat-sen University
ignited global ethical debate about gene edting
technology
2 Sep 2015 Leading UK research councils, including the MRC,  
declared support for using CRISPR-Cas9 and other
genome editing techniques in preclinical research
11 Sep 2015 Hinxton Group issues a statement indicating that  
most of the ethical and moral questions raised
about CRISPR and gene editing have been debated
before
11 Sep 2015 Hinxton Group issues a statement indicating that  
most of the ethical and moral questions raised
about CRISPR and gene editing have been debated
before
15 Sep 2015 UK Nuffield Council on Bioethics held its first  
workshop to identify and define ethical questions
relating to developments in genome editing
research
18 Sep 2015 UK scientists sought license to genetically modify Naikan Crick Institute
human embryos to study the role played by genes in
the first few days of human fertilisation
25 Sep 2015 New protein, Cpf1, found, offering means to simplify Zhang, Zetsche, Broad Institute,
gene editing. Gootenberg, Abudayyeh, Massachusetts Institute
Slaymaker of Technology
5 Oct 2015 CRISPR/Cas9 modified 60 genes in pig embryos in Church Harvard University
first step to create organs suitable for human
transplants
6 Oct 2015 UNESCO’s International Bioethic Committee called  
for ban on genetic editing of human germline
16 Nov 2015 US scientists published a technique for overwriting DiCarlo, Chavez, Dietz, Harvard University, Swiss
changes made by CRISPR/Cas 9 Esvelt, Church Federal Institute of
Technology in Zurich
23 Nov 2015 US scientists genetically modified mosquitos using Gantz, Jasinskiene, University California San
CRISPR/Cas9 to prevent them carrying malaria Tatarenkova, Fazekas, Diego, University of
parasite Macias, Bier, James California Irvine
1 Dec 2015 International Summit on Human Gene Editing met to Baltimore, Doudna, US National Academies of
discuss the scientific, medical, ethical, and Church, Zhang Science, Engineering and
governance issues associated with recent Medicine, US National
advances in human gene-editing research Academy of Medicine,
Chinese Academy of
Sciences, Royal Society
31 Dec 2015 Gene editiing tool, CRISPR, successfully used to Nelson, Gersbach, Hakim, Duke University,
improve muscle function in mouse model of Ousterout, Thakore University of Missouri,
Duchenne muscular dystrophy University of North
Carolina, Massachusetts
Institute of Technology,
Harvard University
6 Jan 2016 US scientists published improved version of Kleinstiver, Pattanayak, Harvard University
CRISPR/Cas 9 with less risk of off-target DNA Prew, Tsai, Nguyen,
breaks Zheng, Joung
1 Feb 2016 UK scientists authorised to genetically modify Niakan Crick Institute
human embryos using CRISPR-Cas 9
16 May 2016 US scientists publish new base editing technique Komor, Kim, Packer, Zuris, Harvard University
offering means to alter genome without needing to Liu
cleave double-stranded DNA or for a donor DNA
template
21 Jun 2016 2016: NIH gives green light for first clinical trial June University of Pennsylvania
using gene editing tool CRISPR/Cas 9 to treat
patients
February 2017 US National Academies of Science and Medicine  
gave green light to proceed with CRISPR in germ-
line experiments
13 Apr 2017 CRISPR shown to be sensitive diagnostic tool for Abudayyeh, Broad Institute,
detecting single target of DNA or RNA molecule Bhattacharyya, Collins, Massachusetts Institute
Daringe, Donghia, Dy, of Technology, Harvard
Essletzbichler, Freije, University, Howard
Hung, Joung, Koonin, Lee, Hughes Medical Institute
Livny, Myhrvold, Regev,
Sabeti, Gootenberg,
Verdine, Zhang
13 May 2017 Research published demonstrating how CRISPR- Yin, Zhang, Qu, Chang, Temple University,
CAS9 can be used to eliminate HIV in infected mice. Putatunda, Xiao, Li, Zhao, University of Pittsburgh,
Dhai, Qin, Mo, Young, Sichuan University
Khalili, Hu
2 Aug 2017 Research published demonstrating possibility of Hong, Marti-Gutierrez, Oregon Health & Science
editing gene defect in pre-implanted human Park, Mitalipov, Kaul, Kim, University, Salk Institute,
embryos for preventing inherited heart disease Amato, Belmonte Center for Genome
Engineering, Seoul
National University, China
National GeneBank,
September 2017 DNA of human embryos edited using CRISPR-Cas9 Fogarty, McCarthy, Francis Crick Instiitute,
to study cause of infertility Snijders, Powell, Cambridge University,
Kubikova, Blakeley, Lea, Oxford University, Seoul
Elder, Wamaitha, Kim, National University
Maciulyte, Kleinjung, Kim,
Wells, Vallier, Bertero,
Turner, Niakan
23 Sep 2017 Chinese researchers report correction of gene linked Liang, Ching, Sun, Xie, Xu, Sun Yat-sen University,
to beta thalassaemia, inherited blood disorder, in Zhang, Xhiong, Ma, Liu, Baylor College of Medicine
human embryos using base editing technique Wang, Fang, Songyang,
Zhou, Huang
25 Oct 2017 New CRISPR technique published for editing RNA Zhang, Cox, Gootenberg, Massachusetts Institute
Abudayyeh, B Franklin, of Technology, University
Kellner, Essletzbichler, of Minnesota
Verdine, Joung, Lander,
Belanto, Voytas, Regev
25 Oct 2017 Base editing improvements announced for CRISPR Gaudelli, Komor, Rees, Massachusetts Institute
technique, providing means to change individual Packer, Badran, Bryson, of Technology, Harvard
chemical letters of DNA without need to cleave DNA Liu University
5 Jan 2018 Researchers identify pre-existing antibodies Charlesworth, Deshpande, Stanford University
targeting CAS9 proteins raising possibility of Dever, Dejene,Gomez-
immune responses undermining utility of CRISPR- Ospina, Mantri, Pavel-
Cas9 for gene therapy Dinu, Camarena,
Weinberg, Porteus
27 Aug 2018 First CRISPR-Cas9 clinical trial launched Vertex Pharmaceuticals,
CRSIPR Therapeutics
24 Nov 2018 First gene-edited babies announced by Chinese Jiankui Southern University of
scientist Science and Technology
of China
14 Dec 2018 New gene modification technique (CRISPRa) makes Matharu, Rattanasopha, University of California
it possible to increase expression of its target gene Tamura, Maliskova, Wang, San Francisco
Bernard, Hardin, Eckalbar,
Vaisse, Ahituv
21 Dec 2018 CRISPR-Cas9 editing helped restore effectiveness Kmiec, Bialk, Wang, Helen F Graham Cancer
of first-line chemotherapies for lung cancer Hanas Center and Research
Institute
23 Jan 2019 CRISPR-Cas9 used to control genetic inheritance in Grunwald, Gntz, University of California
mice Poplawski, Xu, Bier, San Diego
Cooper
30 Jul 2019 World Health Organisation called on countries to  
ban experiments that would lead to more gene-
edited babies
21 Oct 2019 New DNA editing technique called 'prime editing' Anzalone, Randolph, Massachusetts Institute
published Davis, Sousa, Koblan, of Technology, Harvard
Levy, Chen, Wilson, University
Newby, Ranguram, Liu
30 Dec 2019 Chinese scientist convicted for using CRISPR-Cas9 Jiankui Southern University of
in human babies Science and Technology
of China

Science links: Science home | Cancer immunotherapy | DNA | DNA extraction | DNA polymerase | DNA Sequencing |
Epigenetics | Gene therapy | Immune checkpoint inhibitors | Monoclonal antibodies | p53 Gene | PCR | Phage display | Phage
therapy | Plasmid | Recombinant DNA | Restriction enzymes | Stem cells | The human microbiome | Transgenic animals |

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