Since the past few years, there is a term, “CRISPR” that is constantly being thrown around, among various scientific and non-scientific communities. In addition, chances are that you have also come across this term while surfing the internet or scrolling down your social media account.



CRISPR stands for clustered regularly interspaced or interspersed short palindromic repeats. CRISPR normally is a prokaryotic defense system.  Used by Bacteria or archaea to provide it immunity against invading viruses or foreign pathogens. It does it so by first scanning the genome of viruses or foreign pathogen, procuring a piece of its genome.  They do so by either directly cleaving foreign genome or picking up viral DNA or RNA pieces, which viruses left behind them after they invade bacteria.

Bacteria then integrate these pieces into its genome, into a location or locus called CRISPR locus (loci for plural). By this bacteria gains immunity against that specific foreign entity, whose genome they have integrated into themselves.  Now in case of next invasion, bacteria or archaea will active its CRISPR system. It will scan the foreign genome pieces with the help of foreign genomes that it has integrated into itself. If the foreign genome pieces match with that of the invader. CRISPR will recognize it, and cut it, thereby stopping further damage to the prokaryotes.


CRISPR in terms of as a gene-editing tool is known as CRISPR cas9. It is so called because, along with the CRISPR system, CAS proteins are also used, specifically cas9. CAS stands for CRISPR associated proteins.


Now let us talk about when and why CRISPR, as well as CRISPR cas9 gene-editing tool, was discovered and by whom?

Firstly, the clusters of palindromic repeated DNA sequences were discovered in the timeline, which finally ended with the utilization of CRISPR as a gene-editing tool.  From the literature, it is proved that; this discovery of repeated DNA sequences was done at three separate occasions, independently by different groups of researchers.

First, Japanese scientist “Yoshizumi Ishino and his group’’ from the University of Osaka in 1987 reported this repeated DNA sequences, while working with IAP gene. They only reported the existence of these sequences, at that time, they were not able to find the functions of these sequences.

Then in 1993, researchers from the Netherlands, while working on Mycobacterium tuberculosis, reported the presence of repeated sequences, which were intervened by non-repeating sequences, they also saw that these repeated sequences were also present in other Mycobacterium species.

At the same time, a Spanish microbiologist, Francisco Mojica from the University Of Alicante, Spain, also observed the repeated sequences, which were interrupted by non-repeated sequences in archaeal species.

Then in 2001-2002, Francisco Mojica and Ruud Jenson coined the term “CRISPR”. Francisco Mojica further discovered that the intervening sequences between repeated sequences were in fact parts of the bacteriophage genome.

In 2005, three independent research groups showed that the pieces of genomes in intervening sequences were acquired from invading pathogens, invading bacteria and that they all hypothesized that CRISPR may have a role in adaptive immunity of bacteria against viruses and other invading pathogens.

In 2005, CAS9 and PAM( protospacer adjacent motifs) were discovered by Alexander Bolotin from French national institute for agricultural research.  He discovered them while working on Streptococcus thermophiles bacteria. He sequenced it and found some novel sequences in its CRISPR locus, these sequences were for CAS9, a nuclease and also he found out that the spacers or intervening sequences also shared some homology with viral genes. While a set of nucleotides in those spacers, were common across all spacers sequences present at one of the end, these were called PAM or protospacer adjacent motifs, which was used as target recognition by CRISPR.

In March 2006, Eugene Koonin, from the US national center of biotechnology and information.  Presented a hypothesized scheme of how CRISPR works in acquiring foreign genome snippets and then in generating adaptive immunity, while the next year, in 2007,  researchers from France, demonstrated experimentally that how CRISPR works in immunity. Till now we knew how CRISPR worked, but no one knew, the exact details, from here on onwards, scientists began to understand in the details the working of CRISPR.

John var der Oost and his colleagues from the Netherlands in 2008, showed that small RNAs were transcribed from the intervening sequences, and these RNAs were called CRISPR RNAs or crRNA and they act as guides, taking CAS proteins to the target genome.

At this point, all we knew was that CRISPR targets genome and no one knew whether it target DNA or RNA, some even considered that CRISPR is a eukaryotic version of RNA interference (RNAi). Luciano Marraffini and Erik Sontheimer from the Northwestern University, Illinois in 2008, showed that CRISPR target DNA,  later it was found that a different version of CRISPR also targets RNA.

Sylvain Moineau and his colleagues proved CAS9 role in Cleavage in 2010 at the University of Laval, Quebec City, Canada.  Still, one thing was left behind, to tell us the exact working of naturally occurring CRISPR.

Emmanuelle Charpentier and her team in 2010, showed that crRNA does not work alone and another RNA called trans-activating CRISPR RNA or tracrRNA is needed to join with crRNA and make a duplex, which helps in guiding CAS nucleases towards the target.

Now we knew the exact working of naturally occurring CRISPR and how it was discovered.  Now it was time to use CRISPR for gene-editing, the first step was to make it simple because as two RNAs were used to guide CAS proteins, synthesize it. for this two research groups showed independently that by fusing tracrRNA and crRNA, we can make a synthetic single guide RNA or sgRNA, which will guide CAS proteins, towards the target, one of these research groups were of  Virginijus Siksnys and his group.  the second research group was of Emmanuelle Charpentier and Jennifer Doudna.

Finally, it was Feng Zhang, who utilizing his previous knowledge of working on other genome editing tools, modified  CRISPR CAS9 to be used in eukaryotic cells for genome editing.

This was the whole journey of CRISPR first discovery and from CRISPR to becoming CRISPR cas9 genome-editing tool.


Now let us see how CRISPR works naturally, as we know that in nature CRISPR act as an adaptive immunity in bacteria and archaea against viruses and other invaders.

When a virus or foreign particle invades bacteria or archaea, the first step is to acquire spacers or a piece of invader’s genome, to incorporate it into its CRISPR genome and to acquire adaptive immunity against that invader, in case it attacks again.

First CRISPR system activates, and it scans the foreign genome, for a sequence called PAM (protospacer adjacent motif).  This sequence signals CRISPR to use its CAS proteins to cleave foreign genome, and then take it as spacer sequences in CRISPR locus. This step is known as spacer acquisition, in which foreign DNA is taken by bacteria or archaea and stored in its CRISPR loci, hence generating adaptive immunity.  CRISPR utilizing CAS proteins, in spacer acquisition, there are several CAS proteins. Some act as nucleases, cleaving foreign DNA or RNA, some have other functions while some are integrases, which integrate foreign DNA into CRISPR loci.

Then crRNA is transcribed from the spacers present in the CRISPR locus, it is complementary to a specific region in the foreign DNA, it activates CRISPR CAS proteins.

Apart from crRNA, another form of RNA is also made it is called trans-activating CRISPR RNA or tracrRNA which activates crRNA, by binding to it form a duplex. This duplex signals an RNase to cleave it and forms crRNA and tracrRNA hybrid. This hybrid then acts as a guide for CAS nucleases, guiding them towards the target and cleaving the target DNA or RNA.  The target is identified by the PAM sequences and also by the complementary transcript, transcribed from the spacer motifs in the CRISPR locus. This is how CRISPR works in a natural system.


Now let us see how CRISPR works as a genome-editing tool.  Whenever we need to perform gene-editing experiments, our main aim is either to remove or delete a gene or switching off a gene. On the other hand, we may try to turn on a gene by either modifying a gene to make a functional product by inserting some nucleotides or even adding a new gene, but inserting the sequences of a new gene.

First, let us see how CRISPR works in terms of switching off a gene. For this, therefore, a synthetic guide RNA is made, which is complementary to the target gene.  crRNA and tracrRNA are combined to make sgRNA. This sgRNA then guide CAS9 towards the target gene and it cleaves the target gene. When the gene is cleaved. The cell will try to repair it. The cell will repair this cleavage in two basic ways. First, they repair it in a random way. This is called non-homologous end joining or NHEJ, in this process that cell will try to repair any break in the nucleotide sequence. Just as the name suggests in joining the breaks, priority is not given to homology, but the ends are joined in a random way, which as a result gives rise to a very high chance of errors, these errors than in turn make gene non-functional and switch it off.

While the other repairing system used is called homology-directed repair or HDR. This HDR is used in turning on a gene or when inserting a novel gene in a cellular system, for this first we design a sgRNA, for the sequence where we want to add a new gene or turn it on. And then along with sgRNA and other CRISPR machinery, we also add another sequence, which is the sequence of our gene of interest. This sequence is modified in a way, that few new nucleotides are added to it’s 3’ and 5’ ends, these nucleotides are complementary to the region for which we have designed sgRNA.

When the cut is made on the target sequence, cell repair system will get in action. And will try to repair the break via HDR, because we have added the sequence of our gene of interest, which both 3’and 5’ modified. So that the repair system will recognize it and will consider it a part of the genome, which broke off, It will try to join it back and in this way, it will add our gene of interest into a cellular system for expression.

This is how CRISPR/Cas9 is used in gene-editing, both in knocking out a gene and in knocking in a gene.


Now that we know how CRISPR/Cas works,  now let us see what are the components of CRISPR/CAS system. Here we will consider CRISPR/Cas as a genome-editing tool.  And their components are:

  • sgRNA:

a synthetic RNA which is made by combining crRNA and tracrRNA. It guides the CAS nuclease towards the target.

  • Spacer:

It is the sequence which is complementary to the target sequence, it is attached with sgRNA. And after its annealing with the target sequence, then cleaving occurs.

  • CAS proteins:

CRISPR associated proteins,  are a group of proteins, which carry out the main function of CRISPR, there are several CAS proteins, on the basis of which, there are several CRISPR/Cas types. Like for example, CAS9 is used in CRISPR/cas9 system and it is a nuclease which cleaves DNA, similarly, we have CAS13 which cleaves RNA.  

There are also some CAS proteins which are common in almost every CRISPR, and they are CAS1 and CAS2. They both are involved in spacer acquisition and generating adaptive immunity but activating CRISPR and helps in transcribing spacer elements from the CRISPR locus.

  • PAM:

It is a string of three bases, which are needed to present near the target sequence. They act as a recognition site for the CRISPR machinery. After its recognition, spacer sequence binds to the target sequence, followed by the nuclease activity of CAS protein.


Now that we know about what CRISPR is, and how it works, what are its components? Now it is time to see in what ways CRISPR has been utilized so far, as well as what are the potential uses of CRISPR in the future.


let us start with the most obvious application of CRISPR, which is genome-editing we know that about CRISPR prowess in genome editing.  CRISPR is being used in various cell lines and animal models, to find out a cure for single cell genetic diseases. Like sickle cell anemia, cystic fibrosis, hemophilia.

Recently scientists were successful in curing a genetic disease called phenylketonuria, this disease is a metabolic disorder, in which a gene called phenylalanine hydroxylase. When this gene is mutated, over-accumulation of phenylalanine happens. Moreover, this leads to various complications, scientists led by Professor Gerald Schwank at ETH Zurich institute. Where they successfully corrected Phenylketonuria, with the help of CRISPR in mice. Scientists have expressed that soon in near future, we will be able to cure many genetic diseases, mainly metabolic disorders.

Scientists are also working on using CRISPR/cas9 genome editing on various genetic diseases like Huntington’s and Duchene muscular dystrophy.  But apart from such promise being showed by CRISPR, it is still in its developmental phases, and there are several problems with CRISPR, like ethical issues and off-target mutations. Once these issues are solved, CRISPR will truly become a revolutionary medication against genetic diseases.


We know that day by day, this earth is becoming more and more hostile. With global warming and natural resources depletion, are among some of the main problems that we are facing. Now with the application of CRISPR, we can totally change the agricultural sector. Some Korean researchers are now focusing on making Banana resistant to fungal diseases. This is important, because as banana propagate through asexual ways, which makes it prone to diseases, as its genome remains the same, making it impossible to become resistant to diseases naturally.


We are facing a growing epidemic of antibiotic-resistant bacteria due to uncheck and unauthorized use of antibiotics.  Which in turn is helping bacteria to become resistant to it. Now we have bacterial strains like methicillin-resistant Staphylococcus aureus also known as MRSA, Pseudomonas aeruginosa, etc.  Now work is being done on using bacteriophages as vectors to transport CRISPR/Cas machinery inside the bacterium and to use it to kill it instead of relying on antibiotics, in near future, we may be able to cover this problem of resistivity.


CRISPR is a genome-editing tool can also be used in cancer. Because as cancer is developed when some specific gene is mutated.  We can design CRISPR such that it will target only those mutated genes, and correcting them, hence trying to restore the normal function of the gene, hence stopping and even reversing the damage caused by cancer growth.

Currently, clinical trials are carried out in China, where CRISPR in patients with advanced esophagus cancer.  In which T cells are engineered in such a way, as to recognize cancer cells, target them and then kill them. Some researchers are also working on breast cancer, by targeting BRCA 1 and 2 gene and trying to correct them, which when mutated, results in breast cancer.


We can use CRISPR against AIDS, by destroying HIV, either by targeting the viral DNA inside the infected cells or by making the cells resistant to HIV. It has been reported that certain individuals are naturally resistant to HIV infection, due to a mutation in a gene called CCR5.  This gene encodes for a surface protein on immune cells that HIV uses to infect them. We can deliberately mutate this gene to make it resistant. This is currently in early trial stages in animal models and cell lines.


Gene drive is a process by which we propagate some gene in a way that they are inherited more frequently than other genes, in this way not only desirable genes will remain in a population but also it with the passage of time will lead to new speciation.  Currently, CRISPR is used in gene drive against malaria. We know that malaria is spread with the help of mosquitoes. With the help CRISPR, scientists are now targeting a gene called doublesex in Anopheles mosquitoes. When mutated in males it does not cause anything but when present in females. It makes them unable to lay eggs and also makes them unable to suck blood, which in turn means that it prevents the spreading of malaria.


Even though we have found a possible solution for controlling off-target mutation, but still there are several ethical concerns, related to CRISPR. Recently due to claims of He Jiankui, a Chinese scientist, of creating a genetically edited baby via CRISPR. This has made people began to question CRISPR more.  Unless and until there are a strict check and balance system on the use of CRISPR and CRISPR related experiments, we won’t be able to utilize CRISPR to its full potential.

CRISPR Pros and Cons

  • Its pros are that it is fast and cheap as compared to the other gene-editing tools. Moreover, it can design easily.
  • Its cons are that it also has a high percentage of off-target mutations.


We know that with the CRISPR we can cure genetic diseases and make food resistant to diseases. CRISPR/cas9 has many potential uses, which can really help us in several ways, but only when we have gain full command on it, minimizing its negative effects and answering all the ethical concerns related to it, then we will see how revolutionary CRISPR is.

However, CRISPR cas9 is explained in detail by Here, let me tell you what CRISPR or CRISPR cas9 is and why it is such a huge sensation.


Submit a Comment

Your email address will not be published. Required fields are marked *

Share via
Copy link
Powered by Social Snap