Biotechnology Genetics Synthetic Biology

What is CRISPR Cas9?

  • Since the past few years, the term “CRISPR” is constantly creating a buzz among various scientific and non-scientific communities.
  • To edit or modify DNA easily and precisely was never done before, as it was not possible with the previous gene editing technologies such as ‘TALEN’ and ‘ZFN’. Today, it is possible, thanks to CRISPR technology.
  • CRISPR is the most accurate, efficient, fast and cheapest gene-editing tool so far employed in genetic engineering.
  • CRISPR could revolutionize everything from Medicine to Agriculture to energy (biofuels) to Bio-industry.

What is CRISPR?

CRISPR, (pronounced as crisper) stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR is a new gene-editing tool that allows scientists to more precisely than ever before edit or modifies the genome of almost any organism.

Currently, CRISPR is the simplest, versatile and accurate technique in genetic engineering.

What is CRISPR Cas9?

CRISPR in terms of as a gene-editing tool is known as CRISPR-Cas9. It is so called because, along with the CRISPR system, Cas9 proteins are associated.

Cas9 is a DNA nuclease enzyme in nature. It means that CRISPR Cas9 will be used in those experiments which only target DNA.

Components of the CRISPR/Cas System

CRISPR/Cas system includes the following elements:

sgRNA or Synthetic guide RNA

A synthetic RNA which is made by combining crRNA and tracrRNA. It guides the Cas protein (nucleases) towards the target DNA sequences.


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

Cas Proteins

CRISPR associated proteins, are a group of proteins, which carry out the primary function of CRISPR – that is cleaving.

There are several CRISPR Cas proteins, by which, there are several CRISPR/Cas types. For example, Cas9 protein is used in CRISPR/cas9 system, and it is a nuclease which targets DNA. Similarly, Cas13 protein act on RNA targets.

Also, there are other 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 by activating CRISPR/Cas. Moreover, they help in transcribing spacer elements from the CRISPR locus.


CRISPR Pam Sequence is a string of three bases, which are needed to present near the CRISPR target sequences. They act as a recognition site for the CRISPR/Cas9 machinery. After its recognition, spacer sequence binds to the target, followed by the nuclease activity of Cas protein.

Credit: horizon discovery

CRISPR inside Bacterium

In Bacteria, CRISPR plays a crucial role in their immune system against invading viruses or foreign pathogens. Also, there are several other associated proteins called CAS – help its response towards the foreign particle.

When a virus invades a bacterium, CRISPR system is activated. It captures and cut a piece of viral DNA into small fragments with the help of CAS proteins. The virus fragments are then incorporated into a CRISPR locus (loci for plural) of the bacterial genome, hence generating adaptive immunity. This step is known as spacer acquisition.

If the same virus attacks again, the CRISPR loci are transcribed to generate small Guided RNA or gRNA. These RNA guide CAS-protein to the invading viral DNA. Based on sequence complementarity, the Cas-protein binds to its target, rendering it inactive and hence preventing the viral attack. In this way, the bacterium has acquired immunity against a specific virus.

How does CRISPR Work?

CRISPR-Cas9 system involves two key molecules that render changes in the DNA.

Guide RNA

CRISPR Cas 9 works by transcribing CRISPR–loci into RNAs of short sequences – so-called guide RNA (gRNA). This small sequence RNA finds the spots and guide the Cas9 to a target, where some DNA editing or deleting action takes place.

Cas Protein

Once find the DNA target, one amongst the enzymes created by CRISPR system – binds to the DNA and cuts it, rendering it inactive. The Cas proteins are nucleases in nature that cleave DNA at a specific location.

How CRISPR Cas9 Work as a Genome Editing Tool?

Seeing how CRISPR recognizes the target sequence and the way it removes or inserts DNA stretches. This led scientists to think, what if they can control and modify this system, and use it in genetic engineering. Luckily, they succeeded in it, developed a gene editing tool called CRISPR/CAS system.

CRISPR can be easily programmed to target particular sequences of DNA and to edit or remove it at specific locations.

Switching off a gene by CRISPR Cas9

A guide RNA is synthetically designed that is complementary to the target gene. The sgRNA then guide Cas9 towards the target and cleaves it.

Once the gene is cleaved, the cell considered it the DNA damage and will try to repair the cleavage in two fundamental ways. First, the cell fixes it in a random way – called non-homologous end joining or NHEJ. It involves the joining of the cleaved DNA ends in a random way. It results in a very high chance of errors. These errors, in turn, make gene non-functional and switch it off.

Switching on OR inserting a gene by CRISPR cas9

The other repairing system used is called homology-directed repair or HDR.

sgRNA is designed followed by CRISPR Cas9. Also, add sequences of interest- the one need to be inserted or to turn on a gene. The interest sequence is modified, so it’s 3’ and 5’ ends, and make it complementary to the target region.

When the DNA cut is made on the target sequence. Cell repair system recognizes the modified 3’and 5’ sequences is a part of the broke off genome. It will join it back and, in this way, would add our gene of interest into a cellular system.

Comparison of CRISPR Cas9 with other Genome Engineering Technologies

Several genome-editing techniques have been designed to improve gene targeting methods. It includes CRISPR-Cas systems, transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs).

Meanwhile, to precisely edit or modify DNA is difficult, and even not possible with the previous gene editing technologies such as ‘TALEN’ and ‘ZFN’. Today, it is possible, thanks to CRISPR Cas Technology.

  • As compared to other techniques, CRISPR- Cas9 is used as an alternative because it is more effective and customized.
  • Moreover, it is the most accurate, efficient, fast and cheapest gene-editing tool so far employed in genetic engineering.
  • CRISPR system does not require the attachment of separate cleaving enzymes (nucleases) as the other genome editing tools do.
  • Also, it only needs the redesign/construction of CrRNA (sgRNA) to change the specificity of the target DNA.
  • Simultaneously, CRISPR can target multiples genes which are another advantage.

Explore More: Scientists find new and smaller CRISPR gene editor: CasX

Applications of CRISPR

CRISPR is like a “kid in a candy store” – Paul Knoepfler, an associate professor at UC Device School of Medicines.

CRISPR has the potential and can be used in various fields, from Medicines to agriculture in the controlled environment.

According to STAT, there is 1,453% increase in the number of peer-reviewed scientific publication with the CRISPR in title or abstract. This shows us how much effective CRISPR is and how much work is being carried on it.

CRISPR Cas9 for Gene-editing

The most obvious application of CRISPR Cas9 is genome-editing. CRISPR has been used in various cell lines and animal models, to find out a cure for single cell genetic diseases, i.e. sickle cell anemia, cystic fibrosis, hemophilia.

Recently scientists were successful in curing a genetic metabolic disease called phenylketonuria in mice with the help of CRISPR Cas9. A team of scientists led by Professor Gerald Schwank at ETH Zurich institute.

Scientists are optimistic about curing genetic disorders particularly metabolic diseases soon, with the help of CRISPR technology.

Also, researchers used CRISPR to correct several genetic anomalies in human embryos. They had successfully removed a heart disease defect in an embryo using CRISPR.

Moreover, Feng Zhang’s lab at the Broad Institute developed a CRISPR-based diagnostic tool – a SHERLOCK. The new technique could detect tumor DNA in the blood samples, as well as viruses like Zika and dengue.

Also, researchers are working on using CRISPR/cas9 genome editing on various genetic diseases like Huntington’s and Duchene muscular dystrophy.

CRISPR Cas9 Infographic

CRISPR Use in Agricultural Sector

The 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.

Researchers are already looking to use CRISPR and could totally change the agricultural sector. Mainly crops are modified by deleting those genes which can cause mutation in the presence of some environmental factors. Or by inserting some genes, that are resistant to various pests and diseases.

Moreover, giant companies such as Monsanto & DuPont also have begun licensing CRISPR technology. They aim to develop valuable crops with new varieties.

CRISPR as Antibiotic Resistance Solution

We are facing a growing epidemic of antibiotic-resistant bacteria due to uncheck and unauthorized use of antibiotics. Now we have bacterial strains like methicillin-resistant Staphylococcus aureus also known as MRSA, Pseudomonas aeruginosa, etc.

The work is being done on using bacteriophages as vectors to transport CRISPR/Cas machinery inside the bacterium to knockout the antibiotic resistance genes. In the near future, we may be able to cover this problem of resistivity with CRISPR technology.

Use of CRISPR in Cancer

CRISPR is a genome-editing tool can also be used in cancer. As cancer is developed when some specific genes are mutated. The CRISPR could be designed in such a way that it will target only those mutated genes, and correcting them. Hence trying to restore the normal function of the gene.

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 that it recognizes cancer cells, target them and then kill them.

Also, researchers are working on breast cancer, by targeting BRCA 1 and BRCA 2 gene and trying to correct them.

Explore more: Gene Editing Using CRISPR-Cas9 for the Treatment of Cancer!


CRISPR could be used against AIDS, by destroying HIV, either by targeting the viral DNA inside the infected cells or by making the cells resistant to HIV.

CRISPR was successfully used on rats in the controlled environment to remove the HIV-1 virus.

It has been reported that certain individuals are naturally resistant to HIV infection, due to a mutation in a gene called CCR5. The gene encodes for a surface protein on immune cells that HIV uses to infect them. Through CRISPR, we can deliberately mutate this gene to make it resistant. This is currently in early trial stages in animal models and cell lines.

Also Read: Can CRISPR Cure HIV?

CRISPR for Gene Drive

Gene drive involves the propagation of some gene inherited more frequently in such a way that not only desirable genes will remain in a population but also would lead to new speciation.

Currently, CRISPR is used in gene drive against malaria. Scientists are targeting a gene called double sex in Anopheles mosquitoes. When mutated in males it does not cause any effect. While it makes females unable to lay eggs and suck blood which in turn prevents the spreading of malaria.

CRISPR for Storing and Recording data

A group of scientists at Harvard Medical School and Wyss Institute, using CRISPR, to stored and later retrieved data in Escherichia coli. The data was in the form of a clip from “Sallie Gardner at Gallop”, one of earliest motion pictures. Hence paving a way for using CRISPR for storing and recording data.

CRISPR off-target Mutations

Meanwhile, CRISPR is still developing; it may have some flaws. The critical problem with CRISPR Cas9 is unintended mutations.

A study published in Nature, researchers from Columbia University, revealed that CRISPR could cause unintended mutations in the DNA.

The percentage of off-target mutations by CRISPR is ≥50%. These off-target effects are the reasons, CRISPR is still not used in experiments outside animal models and clinical trials.

  • Why off-target effects of CRISPR

    The reasons for off-target mutation is that sometimes there is a mismatch of 3-6 nucleotides of sgRNA. It is enough for the Cas proteins to cleave at a wrong site. And this, as a result, becomes very disastrous.

  • Solution
    Researchers have recently found a solution for these off-target mutations. A study published in Nature has said that with better designing of sgRNA and sound knowledge of the target sequence. We can minimize off-target mutations to such a level that they won’t be a problem anymore.

But as further work is being done on it, positively this problem will be solved in the future.

Ethical Concern with CRISPR

As every technology has two sides – good and bad. Therefore, CRISPR can also be used in a negative way, i.e. the unregulated modification of Human beings. Even though CRISPR is being used to cure cancer and AIDs but still if the use of CRISPR Cas9 in humans is left unchecked, it can create chaos.

Moreover, scientists have found a possible solution for controlling off-target mutation. Meanwhile, there are several ethical concerns.

Recently due to claims of He Jiankui, a Chinese scientist, of creating a genetically edited baby via CRISPR. The claim has made the scientific community began to question CRISPR more.

Unless and until there are a strict check and balance system on the use of CRISPR and related experiments, we won’t be able to utilize CRISPR to its full potential.

Explore More: The Dark Side of CRISPR

Future of CRISPR

But apart from such promise being showed by CRISPR, it is still in its developmental phases. Also, there are several problems associated with CRISPR, like ethical issues and off-target mutations. Once these issues are solved, CRISPR will genuinely become a revolutionary medication against genetic disorders and cancer. Moreover, its potential in agriculture and gene drive and other sectors of life.

Read More: Most Frequently Asked Questions about CRISPR?


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