What is CRISPR?
CRISPR can be used to edit genomes. Researchers can modify DNA sequences and alter gene function with ease. CRISPR has many applications. It can correct genetic defects, treat and prevent the spread of disease, and improve the growth and resilience for crops. The technology has its merits, but there are ethical issues.
CRISPR (pronounced "crisper") can be abbreviated as "CRISPR–Cas9" in popular usage. CRISPRs are specialized DNA stretches. The protein Cas9 (where Cas stands for CRISPR-associated) is an enzyme that functions like a pair molecular scissors capable of cutting DNA strands.
CRISPR technology was inspired by the natural defense mechanisms of bacteria, archaea and other single-celled microorganisms. CRISPR-derivedRNA is a molecular cousin of DNA and various Cas proteins are used by these organisms to foil viruses attacks. The organisms take out the DNA of viruses to foil them and store it in their genome. This DNA can then be used against foreign invaders if they attack again.
CRISPR components can be transferred to other organisms in order to manipulate genes. This is called "gene edit". This process was unknown until 2017 when researchers led by Mikihiro Schibata of Kanazawa University, Japan, and Hiroshi Nakasu, from the University of Tokyo, showed for the first time what CRISPR looks like in action. Live Science previously reported.
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CRISPR's key components
DNA is a double-stranded molecular structure. Its "rungs" consist of either adenine paired thymine or of cytosine and guanine. (Image credit: Shutterstock)
CRISPRs: CRISPR stands for "clusters if regularly interspaced short palendromic repeats". It refers to a region of DNA that is composed of short repeat sequences with "spacers" between them.
Talking about repeats in the DNA code refers to the order of rungs on a spiral ladder. Each rung is composed of two chemical bases that are bound together. A base called "Adenine" links to another called "Thymine" (T), while the base "Guanine" pairs with the base "C".
These bases are found in the CRISPR region in the same order multiple times. According to the Max Planck Institute, these segments form what is known as "palindromic" sequencings. A palindrome is like the word "racecar" in that it reads forward and backward the same way. In a palindromic sequence the bases on one side match the ones on the opposite side of the DNA ladder when they are read in opposite directions.
This is an example of a very simple palindromic sequence.
Side 1 - GATC
Side 2 - CTAG
CRISPR DNA regions contain short palindromic repetitions. Each repeat is bookended by "spacers." These spacers are taken by bacteria from viruses that have attacked them. This allows them to incorporate some viral DNA into their genome. These spacers act as a memory bank, which allows bacteria to identify the viruses in the event of an attack. Spacers can also be thought of as "Wanted" posters. They provide a snapshot of the bad guys to make it easy for them to be identified and brought to justice.
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This process was first demonstrated experimentally by Rodolphe Barrangou, and a Danisco team of researchers. Danisco is a food ingredient company. According to the Joint Genome Institute, part of U.S. Department of Energy, the researchers used Streptococcus temperaturephilus bacteria as their model in a 2007 paper. The bacteria had new spacers in their CRISPR areas after a viral attack. These spacers had a DNA sequence that was identical to the viral genome.
The researchers also modified the spacers, removing them and inserting viral DNA sequences. The researchers were able alter bacteria's resistance against a particular virus attack, which confirmed the role of CRISPRs in controlling bacterial immunity.
CRISPR RNA: CRISPR regions in DNA serve as a bank of viral memories. However, for the stored information to be used elsewhere in the cell it must be copied or "transcribed" into another genetic molecule calledRNA. CRISPRRNA (crRNA), unlike DNA sequences that are kept inside the DNA molecule and can roam around the cell with other proteins, namely the molecular scissors which snip off viruses.
The only difference between DNA and RNA is that it has one strand rather than two. This makes it look like a half-length ladder. One part of the CRISPR is used as a template. Proteins called polymerases then jump in to create an RNA molecular that is "complementary". This means that the bases of both strands would fit together like puzzle pieces. A G in the DNA molecule would be transcribed into RNA as a C.
According to a 2014 Science review, Jennifer Doudna et Emmanuelle Charpentier found that each snippet of CRISPR RNA has a copy of a repeat as well as a spacer from a CRISPR area of DNA. In order to protect bacteria from viruses, the crRNA interacts both with Cas9 and another type of RNA called "trans-activating crRNA" (or tracrRNA).
Cas9: Cas9 is an enzyme that cuts out foreign DNA. Cas9 binds to tracrRNA and crRNA to guide it to the target DNA strand of the virus. The Cas9 will target the crRNA's DNA strands with a complementary 20-nucleotide length. A "nucleotide", which is a DNA building block that only contains one base, is the portion of the DNA the Cas9 will cut.
According to the 2014 Science article, Cas9 uses two distinct regions, or "domains," on its structure. This creates what's known as a double-stranded break.
Cas9 does not just cut any part of a genome. There is an integrated safety mechanism. The "protospacer adjacent motif" or PAMs are short DNA sequences that serve as tags. They sit next to the target sequence. The Cas9 complex won't work if it doesn't find a PAM near its target DNA sequence. According to Nature Biotechnology's 2014 review, this is one reason Cas9 does not attack the CRISPR area in bacteria.
CRISPR as a tool for genome editing
Here's how Crispr gene editing works. (Image credit: ttsz via Getty Images)
Genomes encode a set of messages and instructions in their DNA sequences. Genome editing is the process of changing these sequences and changing the messages they contain. You can insert a cut or break into the DNA to trick a cell's DNA repair mechanisms into making the desired changes. CRISPR-Cas9 provides a means to do so.
Two pivotal research papers published in Science and PNAS in 2012 described how the bacterial CRISPR–Cas9 could be used not only to destroy viruses' DNA but also any other DNA. This allows the natural CRISPR system to be transformed into a simple and programmable tool for genome editing.
Scientists can direct Cas9's ability to cut a particular region of DNA by changing the sequence of crRNA. This binds with a complementary sequence in target DNA. Martin Jinek and colleagues simplified the process further by fusing tracrRNA and crRNA to create one "guideRNA". Genome editing only requires two components: the Cas9 protein and a guideRNA.
"Operationally you create a stretch (20 base pairs) that matches a gene you want to edit," and then one can determine the complementing crRNA sequence, George Church, a Harvard Medical School professor of genetics, explained Live Science. Church stressed the importance of ensuring that the nucleotide sequence only exists in the target gene.
Church explained that the RNA and the protein [Cas9] will then cut like a pair or scissors the DNA at the site. After the DNA has been cut, cells' natural repair mechanisms work to put the DNA back together. At this point, it is possible to make edits to the genome. This can be done in two ways:
One repair method is to glue the two pieces back together, according to the Huntington's Outreach Project of Stanford University. This is known as "nonhomologous ending joining" and can lead to errors in which nucleotides are accidentally deleted or inserted. These could result in mutations that could cause a disruption of a gene.
The second method involves filling the gap with a sequence nucleotides. The cell uses a very short strand of DNA to create a template. Scientists have the ability to provide any DNA template they choose, which allows them to write in any gene or correct a mutation.
Who was the first to discover CRISPR?
Researchers first discovered the nucleotide repeats, and spacers of Crisprs in E. Coli bacteria. This cluster is shown in a scanning electron microscope image. (Image credit: Callista Images/Getty Images)
CRISPRs were first discovered in bacteria by scientists in 1987. However, they didn’t understand the biological significance of the DNA sequences and didn’t call them “CRISPRs” at the time, Quanta Magazine reports. Yoshizumi and his colleagues from Osaka University, Japan, first discovered the nucleotide repeats (and spacers) in Escherichia coli. As technology for genetic analysis improved over the 1990s, other researchers also found CRISPRs within other microbes.
Francisco Mojica, an academic at the University of Alicante, Spain, was the first person to identify the distinctive characteristics of CRISPRs. He found the sequences in 20 microbes according to a 2016 paper in Cell. He initially called the sequences "short frequently spaced repeats" or SRSRs, but later suggested they be named CRISPRs. CRISPR was first used in a 2002 report published in the journal Molecular Microbiology. It was authored by Ruud Jansen, Utrecht University. Mojica had previously corresponded with Jansen.
Scientists discovered Cas genes over the next years and their function. Quanta reported that they also found out that CRISPRs spacers were invasive viruses.
Jennifer Doudna was a professor of biochemistry and biophysics at the University of California Berkeley. She shared the 2020 Nobel Prize for Chemistry with Emmanuelle Charpentier (director of the Max Planck Unit to the Science of Pathogens). Live Science reported that the two scientists were responsible for adapting CRISPR/Cas to make it a useful tool for gene editing.
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Charpentier discovered tracerRNA in his initial research on Streptococcus Pyogenes, a bacteria that causes tonsillitis and sepsis. Charpentier and Doudna collaborated to create the CRISPR/Cas model in a test tube after discovering tracrRNA. The team published their seminal work, in Science, in 2012. They announced that they had successfully simplified the molecular scissors to a gene-editing tool.
Science Magazine reported that Feng Zhang, a biochemist at the Broad Institute, might be eligible for the Nobel Prize for his work on the CRISPR system. Zhang demonstrated the CRISPR system in mammalian cells. Based on this work, Broad Institute was granted the first patent for CRISPR gene editing technology in eukaryotes. Complex cells that have nuclei can hold their DNA.
What has CRISPR looked like?
(Image credit to KEITH CHAMBERS/SCIENCEPHOTO LIBRARY via Getty Images
2013 was the year that Zhang and Church published their first reports on the use of CRISPR/Cas9 to modify human cells in an experiment. The technology is capable of correcting genetic defects in animal models and lab dishes. According to Nature Biotechnology's 2016 review article, Fanconi anemia and cystic fibrosis are just a few examples of these diseases. These studies have opened the door to therapeutic applications for humans.
CRISPR is being used in medicine as a cancer treatment and as a treatment to treat an inherited condition that causes blindness. CRISPR has been used to prevent the spread of Lyme disease, malaria, and other viral diseases to humans. It has also been tested in animal models of HIV to eradicate infected cells, Live Science reported previously. Live Science reported that a Chinese research team attempted to cure HIV in a patient using CRISPR. Although the treatment was not successful, it did not cause any adverse effects.
"I believe the public perception of CRISPR has been very focused on the idea that gene editing can be used clinically to treat disease," Neville Sanjana, a New York Genome Center assistant professor of biology and neuroscience at New York University said. This is a great possibility, but it is just one part of the story.
Related: Ten amazing things scientists did with CRISPR
CRISPR technology can also be used in agriculture and food industries to create probiotic cultures, and to vaccine industrial cultures (yogurt for instance) against viruses. CRISPR technology is being used to increase yield, drought tolerance, and nutritional properties of crops.
Another possible application is the creation of gene drives, which is a genetic engineering technique that increases a trait's chance of passing from one parent to another. This kind of genetic engineering comes from a natural phenomenon where certain versions of genes are more likely be inherited. According to the Wyss Institute, eventually, the trait spreads over generations. According to a 2014 Science report, gene drives could be used for a variety of purposes, including eradicating invasive species and reverse pesticide or herbicide resistance in crops.
BBC News reported that the CRISPR–Cas9 system was used during the COVID-19 pandemic to create various diagnostic tests.
CRISPR was also used recently in the following ways
A team of scientists published research in Science in April 2017 that showed they had developed a CRISPR molecule for detecting strains of viruses in blood serum, urine, and saliva.
Scientists revealed that CRISPR had successfully removed a defect in the heart of an embryo on Aug. 2, 2017.
Researchers announced on Jan. 2, 2018 that they might be able stop fungi from threatening chocolate production by using CRISPR to increase the plant's resistance to disease.
According to BioNews research, CRISPR was upgraded to allow for simultaneous editing of thousands of genes.
The tool has its limitations, but it is versatile.
Church stated that the greatest limitation of CRISPR was its inability to be 100 percent efficient. CRISPR can only edit a small percentage of targeted DNA in an experiment. Doudna et Charpentier (2014) report that gene editing was found in almost half of rice cells that had received the Cas9RNA complex. Other analyses have also shown that editing efficiency can be as high as 80% depending on the target.
Technology can also cause "off-target effects", where DNA is cut at locations other than the target. Unintended mutations can be introduced by this technology. Church also noted that even if the system is on target, it's possible to miss a specific edit. Church called this "genome vandalism."
CRISPR can pose ethical and potential risks
CRISPR technology has many possible applications. This raises questions about the ethics and consequences of manipulating genomes. A number of ethical issues arose in 2018, when He Jiankui (another biophysicist from the Southern University of Science and Technology, Shenzhen) announced that his team had successfully edited the DNA of human embryos and created the first gene-edited baby in the world.
Live Science reported that he was sentenced to three-years imprisonment and fined $3 million ($560,000) for practicing medical without a license, violating Chinese regulations regarding human-assisted reproduction technology, and fabricating ethics review documents. However, his experiments have raised questions about the regulation of CRISPR, particularly given its relative newness.
Related: What do we know about CRISPR safety?
CRISPR is a dangerous technology, and illegal experimentation on human embryos is a severe misuse. Scientists warn that even ethical uses of the technology can pose risks.
Germline editing is the process of making genetic changes to human embryos or reproductive cells, such as sperm or eggs. CRISPR technology has been used to make germline editing. However, these changes can be passed on to future generations. This raises ethical concerns.
Safety risks include variable efficacy, off target effects, and imprecise editing. There is also much still unknown about scientific communities. A 2015 Science article by David Baltimore and a team of scientists, ethicists, and legal experts notes that germline editing can have unintended consequences for future generations. "There are limits to our knowledge about human genetics, gene environment interactions and the pathways and diseases (including the interplay among different conditions or diseases in the same person)".
Oye and his colleagues highlight the ecological impacts of gene drives in the Science article from 2014. Crossbreeding could allow an introduced trait to spread beyond the target population and into other organisms. Gene drives can also decrease the genetic diversity of the target populations, which could impact its ability to live.
Others ethical issues are more complex. Other ethical concerns are more nuanced. What if germline editing becomes a tool for enhancement of human characteristics, instead of a therapeutic tool?
These concerns were addressed by the National Academies of Sciences, Engineering and Medicine, which produced a comprehensive report that included guidelines and recommendations for genome edits.
The National Academies encourage caution when considering germline editing. However, they stress that caution does not necessarily mean prohibition. They suggest that germline editing only be performed on genes that cause serious diseases or when there is no alternative treatment. They also stress the importance of collecting data about the health benefits and risks, and maintaining continuous oversight during clinical trials. The trial organizers should also follow up with participants' families over multiple generations after the trial ends to determine if there have been any changes in the genome.
Additional resources
This animation by TEDEd explains how CRISPR allows scientists to edit DNA.
Learn how CRISPR allows scientists to edit DNA. After winning the Nobel Prize in 2020, Jennifer Doudna delivers her Nobel Lecture.
After winning the 2020 prize. Science Magazine has more information about the ongoing battle for CRISPR patents.
Additional reporting was provided by Alina Bradford (Live Science contributor).
Original publication on Live Science
