43 CRISPR-Cas9 | Summary

CRISPR-Cas9

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.

This image depicts genome editing. It is adapted from a DNA illustration by Spooky Pooka. Credit: Wellcome Images.

Definition

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.

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 at Massachusetts Institute of Technology (MIT) led by Jennifer Doudna and Emmanuelle Charpentier 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 one led by Feng Zhang at MIT-Harvard Broad Institute, one led by George Church at Harvard Medical School and that of Doudna.

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 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.

CRISPR-Cas9: timeline of key events

Date Event People Places
December 1987Discovery of CRISPR mechanismAmemura, Ishino, Makino, Nakata, Shinagawa, Takase, WachiOsaka University
January 18, 2000More 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 2002Dutch 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, SchoulsUtrecht University
August 2005French scientists suggest CRISPR spacer sequences provide cell immunity against phage infection and are a means of degrading DNABolotin, Quinquis, Sorokin, EhrlichInstitut National de la Recherche Agronomique
November 2005American researchers identified new familes of Cas genes which appeared to help in protecting bacteria against invading virusesHaft, Selengut, Mongodin, NelsonThe Institute for Genomic Research
March 2007Experiments 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 virusesBarrangou, Horvath, Fremaux, Deveau, Danisco USA Inc
2008DNA, not RNA, demonstrated to be the molecular target of most CRISPR-Cas systems
February 2008Scientists coin the term 'protospacer' to denote viral sequence that corresponds to a 'spacer' in the CRISPR-Cas9 system
August 2008Scientists characterised the RNA processing pathway in CRISPR systemWageningen University, University of Sheffield, National Institutes of Health
December 2008Scientists published the RNA gene silencing pathway involved in the CRISPR-Cas mechanism University of Georgia, Florida State University
2011Classification of the CRISPR-Cas system is proposed
April 2012DuPont commercialises the first bacterial cultures based on the CRISPR-Cas 9 technology for the production of pizza cheeseDupont
May 2012First patent application submitted for CRISPR-Cas 9 technologyDoudna, CharpentierUniversity of California Berkeley, University of Vienna
August 2012A group of scientists based at Howard Hughes Medical Institute publish a new method that harnesses the CRISPR-Cas9 system for genome editingJinek, Chylinski, Fonfara, Hauer, Doudna, CharpentierUniversity of California Berkeley
December 2012Fast track application for CRISPR-Cas 9 technology submitted to US patent office. ZhangBroad Institute, Massachusetts Institute of Technology
January 2013CRISPR-Cas is used in human genome editing
January 2013CRISPR-Cas is used to edit the genome of a zebrafish
February 2013CRISPR-Cas is used to programme repression and activation of gene transcription
February 2013Scientists from Harvard Medical School announce the successful genetic engineering of human cells using a CRISPR-Cas 9 techniqueMali, Yang, Esvelt, Aach, Guell, DiCarlo, Norville, ChurchHarvard University
March 2013CRISPR-Cas is used in genome editing of Saccharomyces cerevisiae, a yeast species used in wine making, baking and brewing
April 2013Scientists show that CRISPR-Cas mediated gene regulation contributes to the regulation of endogenous bacterial genesWeiss
August 2013CRISPR-Cas used to engineer a rat's genome
August 2013CRISPR-Cas used to engineer plant genomes including rice, wheat, Arabidopsis, tobacco and Sorghum
August 2013Improvements made to the specificity of CRISPR-Cas system
March 2014First patent awarded for CRISPR-Cas9 technology to be used for a wide range of applicationsZhangBroad Institute, Massachusetts Institute of Technology
March 2015Scientists suggest the use of CRISPR/Cas9 in conjunction with stem cells to provide human organs from transgenic pigsFeng, Dai, Mou, Cooper, Shi, Cai Shenzhen University, University of Pittsburgh Medical Center, Guangxi University
March 26, 2015US scientists call for a voluntary worldwide moratorium on the use of genome editing tools to modify human reproductive cellsLamphier, Urnov
April 2015National Institutes of Health declares it will not fund any use of genome editing technologies in human embryos
April 22, 2015UK Nuffield Council on Bioethics launched a new working group to look into institutional, national and international policies and provisions relevant to genome editing
May 2015Chinese 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, ZhangSun Yat-sen University
September 2015UK 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, 2015Hinxton 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, 2015UK Nuffield Council on Bioethics held its first workshop to identify and define ethical questions relating to developments in genome editing research
September 18, 2015UK scientists seek license to genetically modify human embryos to study the role played by genes in the first few days of human fertilisationNaikanCrick Institute
September 25, 2015New protein, Cpf1, found which could simplify genome editing. Zhang, Zetsche, Gootenberg, Abudayyeh, SlaymakerBroad Institute, Massachusetts Institute of Technology
October 5, 2015US Scientists report use of CRISPR to modify 60 genes in pig embryos in first step to create pig organs suitable for use in humansChurchHarvard University
October 6, 2015UNESCO’s International Bioethic Committee called for ban on genetic editing of human germline
November 16, 2015US Scientists publish a technique for overwriting changes made by CRISPR/Cas 9DiCarlo, Chavez, Dietz, Esvelt, ChurchHarvard University, Swiss Federal Institute of Technology in Zurich
November 23, 2015US scientists report successful use of CRISPR/Cas9 to genetically modify mosquitoes to make them poor hosts for malaria parasiteGantz, Jasinskiene, Tatarenkova, Fazekas, Macias, Bier, JamesUniversity 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 researchBaltimore, Doudna, Church, ZhangUS National Academies of Science, Engineering and Medicine, US National Academy of Medicine, Chinese Academy of Sciences, Royal Society
December 31, 2015CRISPR 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 patientsNelson, Gersbach, Hakim, Ousterout, ThakoreDuke University, University of Missouri, University of North Carolina, Massachusetts Institute of Technology, Harvard University
January 6, 2016Harvard scientists publish improved version of CRISPR/Cas 9 with less risk of off-target DNA breaksKleinstiver, Pattanayak, Prew, Tsai, Nguyen, Zheng, JoungHarvard University
February 1, 2016UK scientists authorised to genetically modify human embryos using CRISPR-Cas 9NiakanCrick Institute

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