One of the most exciting recent developments in genetic engineering is CRISPR-Cas9 (CRISPR).
CRISPR derives its name from “clustered regularly interspaced short palindromic repeats,” genomic sequences that microbes use to defend themselves against viral attacks.
Along with CRISPR associated (Cas) proteins, bacteria use the sequences to recognize and disarm future invading viruses. Scientists have adopted this system for use in genetic engineering.
CRISPR-Cas technology allows scientists to edit genes and manipulate gene expression with a level of ease that was not possible using other methods.
Importantly, it also allows researchers to edit genes within living organisms, a fact that supports the use of CRISPR-Cas in a far-reaching range of applications from basic research to the development of novel therapies and other biotechnology products.
Humans have complex immune systems that involve the coordinated activities of multiple cell types, organs, and signaling systems to recognize and respond to active infections. Prokaryotes — bacteria and archaea — also have a form of adaptive immunity that allows them to recognize and respond to viral infections.
Bacterial Chromosome A CRISPR region within a microbial genome.
CRISPR enables gene editing on an unprecedented scale
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
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.
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.
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.
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
What is CRISPR? The revolutionary gene-editing tech explained
Until very recently if you wanted to create, say, a drought-resistant corn plant, your options were extremely limited. You could opt for selective breeding, try bombarding seeds with radiation in the hope of inducing a favourable change, or else opt to insert a snippet of DNA from another organism entirely.
But these approaches were long-winded, imprecise or expensive – and sometimes all three at the same time. Enter CRISPR. Precise and inexpensive to produce, this small molecule can be programmed to edit the DNA of organisms right down to specific genes.
The development of cheap, relatively easy gene-editing has opened up a smorgasbord of new scientific possibilities. In the US, CRISPR-edited long-life mushrooms have already been approved by authorities while elsewhere researchers are toying with the idea creating spicy tomatoes and peach-flavoured strawberries.
But the game-changing technology could have the biggest impact when it comes to human health.
If we could edit out the troublesome mutations that cause genetic diseases – such as haemophilia and sickle-cell anaemia – we could put an end to them altogether.
The path for human gene-editing is littered with controversies and tough ethical dilemmas, however, as the news in late 2018 that – against all ethical guidance – a Chinese scientist had secretly created the first gene-edited babies.
Here’s everything you need to know about the complex and sometimes controversial technology driving the gene-editing revolution.
What is CRISPR?
CRISPR evolved as a way for some species of bacteria to defend themselves against viral invaders.
Each time they faced a new virus, bacteria would capture snippets of DNA from that virus’ genome and create a copy to store in its own DNA.
“They gather a set of sequences that they’ve been exposed to,” says Malcolm White, a biologist at the University of St Andrews, “these [bacteria] essentially carry a little library in their genome.”
What are genome editing and CRISPR-Cas9?
Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism's DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed.
A recent one is known as CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9.
The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other existing genome editing methods.
CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays.
The CRISPR arrays allow the bacteria to “remember” the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses' DNA.
The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.
The CRISPR-Cas9 system works similarly in the lab. Researchers create a small piece of RNA with a short “guide” sequence that attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme.
As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used.
Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.
Genome editing is of great interest in the prevention and treatment of human diseases. Currently, most research on genome editing is done to understand diseases using cells and animal models. Scientists are still working to determine whether this approach is safe and effective for use in people.
It is being explored in research on a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease.
It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.
How does CRISPR allow us to edit our DNA?
How does CRISPR allow us to edit our [email protected]:16:46+00:00
- This is a dynamic, biological process with many moving parts.
- In my opinion, this type of dynamic activity is best understood with a video.
- First watch the movie, then I’ll further discuss key aspects to CRISPR below…
Jennifer is a professor at UC Berkeley.
She is widely regarded as the main inventor of CRISPR.
In this TED talk, Jennifer does a great job explaining the basics of this gene editing tool.
In this next video, Jennifer describes what happens after CRISPR directs a double stranded break in DNA.
In summary, there are two possible CRISPR options:
- If you want to disrupt a gene, you can rely on the cell’s natural system to repair the double strand break. This repair mechanism will often introduce a mutation in your gene of interest. This is helpful if you want to alter or remove the gene.
- If you want to repair a mutated gene, you can insert repair template with the CRISPR tools. This repair template is a chance for scientists to introduce new sections of DNA. The new DNA will insert at the location of the CAS-9 cut.
Scientists discovered this gene editing system through their work with bacteria. Bacteria use CRISPR as a natural mechanism to help defend against viruses. Part of being a bacteria means you are often under assault from viruses.
CRISPR is a cluster of DNA on the bacterial genome that can be deployed when a virus invades a bacteria. This DNA region will activate and join forces with a CAS-9 protein.
In doing so – the bacteria will slice up the viral DNA before it infects the bacterial genome.
In summary, CRISPR evolved naturally within bacteria to help the bacteria fight viruses.
Did one person discover CRISPR?
Scientific discoveries usually occur because of many small contributions from lots of people. CRISPR is no different.
Many scientists observed that CRISPR is a natural defense system in bacteria. In 1993, Francisco Mojica was the first researcher to characterize what is now called CRISPR.
However, Professor Doudna and her colleagues are credited with discovering how CRISPR can be used to direct genome edits in bacteria. These scientists modified the system to make it more user-friendly for other scientists.
In 2012, the Doudna and Charpentier labs simplified the guide RNA component (combining 2 RNA elements into 1). They then confirmed that CRISPR could be used to direct double stranded DNA cuts at any desired location. In 2013, Dr.
Feng Zhang (Broad Institute of MIT and Harvard) is credited with the key innovation of adjusting CRISPR so that it can direct DNA edits to mammalian cells (i.e. humans).
As you would imagine, there is currently a protracted legal battle occurring for the patent rights for CRISPR.
2019 update: A federal appeals court has, once again, ruled in favor of the Broad Institute. This ruling confirms an earlier US patent board decision, which states that Feng Zhang’s work did not “interfere” with patents at UC Berkeley. The Broad issued a statement reiterating their successful stance.
What is CRISPR?
CRISPR is a technology that can be used to edit genes and, as such, will likely change the world.
The essence of CRISPR is simple: it’s a way of finding a specific bit of DNA inside a cell. After that, the next step in CRISPR gene editing is usually to alter that piece of DNA. However, CRISPR has also been adapted to do other things too, such as turning genes on or off without altering their sequence.
There were ways to edit the genomes of some plants and animals before the CRISPR method was unveiled in 2012 but it took years and cost hundreds of thousands of dollars. CRISPR has made it cheap and easy.
CRISPR is already widely used for scientific research, and in the not too distant future many of the plants and animals in our farms, gardens or homes may have been altered with CRISPR. In fact, some people already are eating CRISPRed food.
CRISPR technology also has the potential to transform medicine, enabling us to not only treat but also prevent many diseases. We may even decide to use it to change the genomes of our children. An attempt to do this in China has been condemned as premature and unethical, but some think it could benefit children in the future.
CRISPR is being used for all kinds of other purposes too, from fingerprinting cells and logging what happens inside them to directing evolution and creating gene drives.
The key to CRISPR is the many flavours of “Cas” proteins found in bacteria, where they help defend against viruses. The Cas9 protein is the most widely used by scientists. This protein can easily be programmed to find and bind to almost any desired target sequence, simply by giving it a piece of RNA to guide it in its search.
When the CRISPR Cas9 protein is added to a cell along with a piece of guide RNA, the Cas9 protein hooks up with the guide RNA and then moves along the strands of DNA until it finds and binds to a 20-DNA-letter long sequence that matches part of the guide RNA sequence. That’s impressive, given that the DNA packed into each of our cells has six billion letters and is two metres long.
What happens next can vary. The standard Cas9 protein cuts the DNA at the target. When the cut is repaired, mutations are introduced that usually disable a gene. This is by far the most common use of CRISPR. It’s called genome editing – or gene editing – but usually the results are not as precise as that term implies.
CRISPR can also be used to make precise changes such as replacing faulty genes – true genome editing – but this is far more difficult.
Customised Cas proteins have been created that do not cut DNA or alter it in any way, but merely turn genes on or off: CRISPRa and CRISPRi respectively. Yet others, called base editors, change one letter of the DNA code to another.
So why do we call it CRISPR? Cas proteins are used by bacteria to destroy viral DNA. They add bits of viral DNA to their own genome to guide the Cas proteins, and the odd patterns of these bits of DNA are what gave CRISPR its name: clustered regularly interspaced short palindromic repeats. Michael Le Page
Crispr: is it a good idea to ‘upgrade’ our DNA?
Last year Tony Perry made mice that would have been brown-furred grow up white instead.
That Perry, a molecular embryologist at the University of Bath, tweaked their coat colour isn’t new – scientists have been making so-called knock-out mice, in which certain genes are disabled, since the technique was invented in 1989. It is a long and cumbersome procedure that involves combining pieces of DNA in embryonic stem cells and mouse breeding.
But Perry, who published his study in December, didn’t use this method. Instead he used a new genome-editing technology that has been taking the scientific world by storm since it was first developed from the bacterial immune system in 2012, and shown to work in human cells in 2013.
The powerful tool, known as Crispr, allows the precise and easy manipulation of the DNA in the nucleus of any cell.
Make the manipulations in sperm, egg or a one-cell embryo, which is just about to start replicating its DNA, and they can become permanently sealed in the so-called germ line, to be inherited by future generations. Using the procedure on the germ line, Perry inactivated a key gene for mouse coat colour.
But Perry’s work added a unique flourish. He did the editing not in a one-cell mouse embryo – which is how most animal germ-line editing by Crispr has been done to date – but earlier, during the process of fertilisation, by injecting the Crispr components and the mouse sperm into the mouse egg at the same time.
It is the same technique – intracytoplasmic sperm injection (ICSI) – widely used in IVF. And it worked. “This or analogous approaches may one day enable human genome targeting or editing during very early development,” notes the paper published in the journal Scientific Reports.
If human germ-line editing were ever to be used clinically, incorporating Crispr into the ICSI phase of IVF is how it might be.
That prospect tantalises Perry because it raises the possibility of generating offspring that carry either no risk or a reduced risk of some genetic diseases. Perry suggests it might one day be possible to correct a harmful mutation in the BRCA1 gene and stop someone inheriting that predisposition to breast cancer. “You will be able to eradicate it from your descendants,” he says.
Crispr can be thought of as a pair of molecular scissors guided by a satnav. The scissors are a DNA-cutting enzyme; they snip at a precise point in the cell’s DNA specified by researchers using a customised guide molecule, a single short piece of RNA, DNA’s chemical cousin. The DNA-cutting enzyme is known as Cas9, hence the technique is often written Crispr-Cas9.
Since the 70s there has been a consensus that human germ-line modification is off bounds
The genome editing occurs as the cell rushes to naturally repair the break made by the scissors.
The cell’s repair often isn’t exact enough for the gene that has been cut to keep working and the gene is effectively knocked out or turned off.
More complex to accomplish, though more precise, genes can also be corrected or whole new genes added if a new piece of DNA is included along with the Crispr machinery. It becomes patched in during the cellular repair process.
Germ-line genome editing is highly controversial, even for medical purposes.
Since the development of genetic engineering in the 70s there has been a “fairly undisturbed” consensus that human germ-line genetic modification – with the worries it raises about “playing God” and “designer babies” – is off bounds, says Peter Mills, assistant director of the UK Nuffield Council on Bioethics and the council’s lead on genome editing. According to Unesco’s Universal Declaration on the Human Genome and Human Rights, germ-line interventions “could be contrary to human dignity”.
The UK government’s decision this February to allow mitochondrial substitution in the clinic to prevent embryos developing with mitochondrial diseases, a form of germ-line therapy, was premised on the basis that the small amount of DNA mitochondria contain is found outside the cell nucleus. There is no modification to the DNA in the nucleus, the real stuff that makes us who we are.
Crispr co-developer Jennifer Doudna.
But there has also never before been a tool shown to be sufficiently effective or reliable enough to seriously consider conducting human genetic engineering.
Other precise genome editing tools – Zinc Finger Nuclease (ZFN) and Talen – have been around longer but Crispr is much easier and cheaper to use, accelerating the science and potential applications.
“The revolution is in access,” says Dana Carroll, a biochemist at the University of Utah who works on improving the tools. “The technology is developing very rapidly.”
The potential for modifying humans weighs on the minds of some scientists, particularly since the publication last month of a Chinese paper which reported using Crispr to genome-edit human embryos for the first time. (The aim was to correct the gene defect that causes the blood disorder beta-thalassaemia and non-viable embryos, which couldn’t have resulted in live birth, were used.)
In an opinion piece published in the journal Science in March, a group of US scientists led by the Crispr co-developer Jennifer Doudna from the University of California, Berkeley recommended steps be taken to “strongly discourage” any attempts at germ-line modification therapy that would produce genome-edited humans while the social and ethical implications are considered. They are calling for an international meeting to consider the appropriate way forward for use. “Lets do this now before the technology is applied in ways which people might feel very uncomfortable about,” says Doudna.
A second group writing in the journal Nature went further, suggesting a moratorium on research where human germ cells are edited for fear of where it might lead.
The backlash should a modified human be born could, they warn, harm work to develop genome-editing therapy for adults and children where the modifications aren’t passed on.
“People might not prise apart the nuances,” says Edward Lanphier, the president and CEO of California-based Sangamo Biosciences, which is pursuing that work.