Gene Editing: CRISPR Cas9: Revolution in genetic engineering

Principle of CRISPR

Gene Editing
  1. While R-DNA technology developed with our understanding of how virus affected bacteria, CRISPR developed out of our understanding of how bacteria developed resistance to this virus.
  2. CRISPR is nothing but the mechanism of bacterial immune system.

Working of CRISPR

  1. When a virus infects bacteria, after few generations bacterial immune system develops a way to fight it back.
  2. The bacterial immune system simply cuts the viral genome using an enzyme (restriction enzyme). In addition, the bacterial immune system keeps a memory of the viral genome in its own genome to ward off future attacks.
  3. This viral genome stored by the bacteria acts as a guide to bacteria during the next viral attack.
  4. So it knows precisely where to cut.
  5. The bacteria had repeat patterns of the viral genome interspersed by a 20-base long DNA.
  6. It also read the same backwards and forwards.
  7. This is why we call the memory of viral genome in bacteria as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
  8. Next time the virus attacks bacteria uses this memory, make a RNA molecule that acts as a guide and Cas 9 enzymes uses this guide RNA to cut the viral genome.
  9. In short, while R-DNA involved cutting of genome blindly (like a shredder), CRISPR Cas 9 enables cutting very precisely.
  10. CRISPR sequence acts like a torch for Cas 9 enzyme that acts like scissors.
  11. In gene editing we use this technique to correct a harmful mutation, insert a missing gene, augment the genome with a new gene in living organisms.
  12. Mimicking the bacterial immune system, we can prepare an RNA-guide molecule in our laboratory which can act as torch for Cas9 enzyme to introduce cuts precisely in the genome.

CRISP-based Gene editing

  1. Once the RNA-guided Cas9 enzyme cuts the DNA at a specific site, we could make the following changes
    1. Inserting a new sequence
    1. Deleting the sequence
    1. Modifying the sequence
  2. In order to do so the cell can use its natural DNA repair mechanisms (DNA polymerase) to insert a nucleotide or introduce a new piece of foreign DNA.
  3. Based on this criterion of using foreign genome v/s altering native genome in gene editing the end product could be transgenic or simply genetically modified.


  • In CRISPR editing, mutations are corrected by cutting the double strand of the DNA. This could result in some unintended cuts.
  • As a result, it can cut only large portions of genome and not single letter changes.
  • Additionally, it can induce unintended off-the-target mutations or incomplete edits in some places.

Advancements in gene editing systems

Base editing

  1. Advanced form of CRISPR, which is suitable for single letter editing.
  2. While Cas9 cuts double stranded DNA target, base editing uses an enzyme to rearrange some atoms in the base molecule of the DNA, thereby altering it.
  3. It involves rewriting the DNA instead of cutting the target sequence and adding new base molecules.
  4. This makes it suitable for single-letter mutations.

Prime editing

  1. Another advanced form of CRISPR, but unlike crispr which chops DNA in half, prime editing nicks it and writes a new section of DNA in the specified region similar to base editing,
  2. Though it is similar to base editing, it includes an additional enzyme, a reverse transcriptase, to copy and paste new DNA sequences into the genome.
  3. Thus, it is suitable for precise insertions, deletions, and augmentation of genome.
  4. While base-editing is suitable for single-letter modifications, prime editing is suitable for more extensive large edits.

Regulation of gene editing in agriculture in India: (SDN1 and SDN2)

  • India does not have a law to regulate gene edited organisms. In contrast, we regulate Genetically Modified Organisms including GM crops and GM food.
  • Rules for the Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms/Genetically Engineered Organisms or Cells, 1989” notified under the Environment Protection Act, 1986, regulate genetically modified organisms.
  • There is no explicit mention of the term gene editing.
  • Recently an amendment was introduced to the aforesaid regulation in order to encourage gene editing in agriculture.
  • Accordingly, gene editing techniques using native genome is allowed in India. However, gene editing technique using foreign genome is not allowed.
  • The gene editing techniques called SDN 1 and 2 (Site-Directed Nuclease) fall under the former category are allowed under the Rules under EPA, 1986.
  • Other techniques like SDN 3, 4 and 6 fall under the latter category and therefore are not allowed in India.
  • In effect, transgenic crops using gene editing are not allowed under the Environment Protection Act.
  • Note that ICAR is using SDN 1 and 2 to produce rice varieties which are drought-resistant, salinity-resistant and high-yielding.

Scope and application

CRISPR system has emerged as panacea for genetic engineering in the recent times with significant contribution to agriculture and medicine. Following are some landmark achievements of CRISPR system.

Genetic Engineering: Recent advancements

What all can CRISPR system do?

Gene editing: DNA and RNA both

  • Both coding and non-coding parts including RNAs (gene expression)Initially only DNA was the target, recently RNA is also being modified using CRISPR system.
  • Conventionally could change a section of DNA (large cuts)Now with base editing suitable for single-letter mutations.


  • Gene therapies not only to correct a defective gene but also silence a gene from expressing or make a gene that was otherwise not expressing to express.

Molecular diagnosis

  • In addition to editing, CRISPR can be used to detect single target DNA or RNA molecule (CRISPR Cas13)
  • This makes it a sensitive diagnostic tool to detect mutations.


  • A biological detective that uses CRISPR-Cas13 to detect RNA sequences associated with diseases like Zika virus and Dengue virus.

CRISPR in Gene therapy

  • CRISPR is an emerging choice for gene therapy given its ability to accurately target defective gene, add a missing gene, alter the defective gene or even augment the genome with a new gene.
  • Besides, scientists have demonstrated administration of CRISPR-based gene therapy both in-vivo (directly in human body) and ex-vivo (from lab culture to human body).
  • CRISPR-based gene therapy is most suitable in mono-genetic diseases in which we can identify a single gene responsible for the disease trait.
  • Examples of such diseases include Blood-related disorders like sickle-cell anaemia, beta thalassemia, Haemophilia etc. Corneal diseases Degenerative neurological diseases Immunological diseases like HIV, bubble boy syndrome etc. Immune-therapy for cancer (CAR T-cell therapy)Skin diseases. Though in a nascent stage it is making rapid strides. Following are some of the recent advancements
  • Scientists have demonstrated how CRISPR-CAS9 can be used to eliminate HIV in infected mice.
  • Gene editing tried in mouse to correct genes involved in muscular dystrophy. Gene editing has been carried out inside the human body (in-vivo) for the 1st time to treat Hunter’s syndrome.
  • US has approved CAR T-cell therapy which involves modifying immune cells to attack cancer cells in case Leukaemia.
  • In 2021 Department of Biotechnology supported 1st  CAR-T cell therapy was conducted in India.
  • Clinical trials for gene therapy for beta thalassemia and sickle cell anaemia is already showing promising results.
  • 1st in-vivo administration of gene therapy to modify  photoreceptor cells in the eye to treat blindness was done in 2020.

CRISPR in tissue engineering and organ transplantation

CRISPR and Cell Therapy

  • CRISPR is increased being used in tissue engineering to genetically modify pig cells to make them suitable for growing human organs in them.
  • Note: Stem cells from humans can be introduced in pigs to grow organs in them as they have a faster life cycle.
  • Problem was a section of pig genome was known to cause cancer which acted as a major hurdle in organ transplantation from pigs to humans.
  • These cancer-causing genes are editing using CRISPR to silence them.

CRISPR in agriculture: GM Crops

  • To engineer crops to increase their nutritional value, pest resistance, drought tolerance etc.
  • Two techniques namely SDN 1 and 2 technique for gene editing
  • ICAR is using it to produce rice varieties which are drought-resistant, salinity-resistant and high-yielding

CRISPR in climate-smart agriculture

  • CRISPR is used in plants to increase their photosynthetic efficiency by upto 25%. Eg: tobacco plants.


  • Besides CRISPR system is being used to increase the carbon fixing property in algae (remember CCUS technologies) by increasing its photosynthetic efficiency.

CRISPR and biofuels

  • CRISPR is used to genetically modify microbes like yeast to improve its efficiency of fermentation and producing ethanol at a faster rate.
  • CRISPR is used to genetically modify methanogens to improve their performance in biogas production
  • Note: Methanogens are microbes that produce methane as a by-product of their metabolism.

GM food

Lab-grown meat

  • Cell-based meat that is produced by culturing
  • cells in a lab instead of livestock rearing practices (major factor in climate change)
  • However the challenge is cell-based meat does not have a suitable texture and flavour.
  • CRISPR systems are used to genetically modify cells to produce proteins responsible for texture and flavour.

Clean Meat Project: India

  • The Clean Meat project will be taken up by CCMB and National Research Centre on Meat of ICAR.
  • Gene edited lab-cultured meat to augment its nutritional content.

Industrial fermentation

CRISPR is used to genetically modify microbes like yeast used in wine making, baking and brewing to improve its efficiency of fermentation.

Gene editing and embryonic cells  

Moratorium on gene editing of embryonic cells

  • In 2018 a Chinese scientist announced the birth of 1st gene-edited babies in the world named Lulu and Nana.
  • After this WHO urged countries to ban experiments that would lead to more gene-edited babies.
  • Gene editing the embryonic cells are banned across the world.
  • In 2019 ICMR issued the National Guidelines for Gene Therapy which also bans gene-editing of embryonic cells.

Note: The Chinese scientist modified CCR5 gene on the embryonic cells of the couples to make them resistant to the HIV virus.

  • CCR5 is a gene that codes for receptors in our immune cells which HIV uses like a gateway to get inside the cell.
  • However CCR5 gene is not just associated with HIV, it may also play an important role in other diseases about which we do not know much.

Research on embryonic cells

  • While there is a world-wide moratorium on gene editing of embryonic cells, research is allowed on embryonic cells that are less than 14-days old.
  • This is needed to understand the nature of many inherited diseases.

Accordingly following research is going on:

  • Pre-implanted human embryos are being tested for understanding inherited heart disease.
  • Genome of embryonic cells are edited using CRISPR-Cas9 to study cause of infertility
  • Research on gene linked to beta thalassaemia, inherited blood disorder, in human embryos using base editing technique is being carried.

CRISPR and Gene Drive: Global fight for Malaria

What is Gene Drive?

  • Gene Drive is the use of gene editing technique to alter the law of inheritance to pass on a particular genetic trait from one generation to next faster than normal.
  • Under normal law of inheritance, a specific trait from an organism has 50/50 chance to be passed. (because the offspring can express either mother’s version or father’s version)’
  • This is achieved editing a particular gene in a way that it can copy and paste itself into its corresponding location on the other chromosome, instead of the 50/50 inheritance pattern that occurs naturally.
  • This copying and pasting occurs during the production of reproductive cells (sperm or egg), resulting in a higher probability that the gene will be passed down to the next generation.
  • Then CRiSPR is used to edit a gene called ‘doublesex’ in female mosquitoes which are the main transmitters of malaria.
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  • When the female mosquitoes inherit two copies of the disrupted gene, they develop like males and are unable to bite or lay eggs.
  • This genetic tweak of double-sex gene follows gene drive inheritance.
  • With this in 8 generations female mosquitoes were completely eliminated
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