The cloud-nine of CRISPR

Review

Debjyoti Ghosh

  The article gives a brief overview of one of the most path-breaking discoveries interdisciplinary sciences in the 21st century - the CRISPR-Cas9 system.



  This year, the Nobel Prize in Chemistry is awarded for discovering gene technology's one of the most invaluable tools – the CRISPR genetic scissors. Prof. Emmanuelle Charpentier from Max Planck Institute of Infection Biology, Berlin and Prof. Jennifer Doudna from the University of California, Berkeley published this revolutionary finding in 2012.1

Prof. Emanuelle Charpentier (left) & Prof. Jennifer Doudna (right) Prof. Emanuelle Charpentier (left) & Prof. Jennifer Doudna (right)

  CRISPR is a powerful RNA-guided DNA2 targeting platform for genome editing, imaging and alteration at the transcription level. This technology can precisely manipulate any genomic sequence identified by a small region of guide RNA. The outcome of the process is a huge boon in terms of the difficulty in understanding gene functions responsible for disease developments. The technology also provides scope to figure out rectification of the disease-causing changes inflicted by the viral attack on the host system such as bacteria etc.

Artist’s impression of bacteriophages attacking a bacteria Artist’s impression of bacteriophages attacking a bacteria. The singular globular green coloured structure is bacteria where the ones in yellow are viruses. Bacteria are unicellular microscopic organisms whereas viruses that attack them are called bacteriophages.
Source: Pintereset

  Many bacteria such as Streptococcus, Staphylococcus, and Saccharomyces, etc. and similar organisms like archaea have their immune systems encoded by CRISPR loci and the CRISPR-associated (Cas) genes to provide immunity against invading virus called bacteriophage and transfer of foreign DNA molecules called plasmids.

All hereditary information is stored in cells in the form of genomes. Cells contain chromosomes which in turn carry long fibres called chromatin made up of DNA (building blocks called nucleotides, which are organic macromolecules) All hereditary information is stored in cells in the form of genomes. Cells contain chromosomes which in turn carry long fibres called chromatin made up of DNA (building blocks called nucleotides, which are organic macromolecules).
Source: iStock
Picture showing molecules that make up DNA and RNA - the genetic material guiding all life-forms Picture showing molecules that make up DNA and RNA - the genetic material guiding all life-forms
Source: iStock

  All hereditary information in a cell is contained in the genome. The genome consists of DNA and RNA. DNAs get converted into RNA through a process called transcription. RNAs make different types of proteins by the translation process. DNA and RNAs are giant organic macromolecules made up of nucleotides. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is a specialized region of DNA with two distinct characteristics – the presence of repeated sequences of nucleotides and interlaying spacers.

  Spacers are the genetic information of the invading bacteriophages.3 Once the spacers are inserted in the CRISPR region of DNA, transcription begins and eventually forms CRISPR RNA (crRNA). The resultant crRNA guides a DNA cleaving protein called nuclease (Cas9) to cut down the invading viral DNA sequence, thereby preventing further infection. Interestingly, Cas9 doesn't cleave randomly. Specific motifs called PAMs (Protospacer adjacent motifs) serve as indicators. They sit adjacent to the target DNA sequences, thereby helping the Cas9 nuclease detect and chop off the intended target.4 Such efficient cleaving features of the CRISPR-Cas9 system raised the prospect of using this system as an efficient and accurate genome editing tool.

  The first-ever demonstration of this technology was seen by Prof Rodolphe Barrangou and a team at Danisco, a food ingredients company in 2007 using Streptococcus thermophilus as the model system (an extensively used bacteria in dairy industries).5.

  In 2012, scientists demonstrated that crRNAs could be constructed to guide the Cas nucleases to any DNA sequences. The Cas9 nuclease was the first and the most prevalent of all Cas nucleases implicated. Subsequently, the scientists coined the defense mechanism as the 'CRISPR-associated protein 9 (Cas9) system.

The CRISPR processEdited: Arunima Vadlamannati

  Some of the recent findings involving the CRISPR system tell us about the impact of the amazing discovery. Now, identifying viral strains in blood serum, urine and saliva6; removing a heart disease in an embryo7; editing thousands of genes at once in a single experiment is possible8. Altering the genome of crops to make them resilient against droughts is happening through CRISPR technology9. It would certainly have taken several more years down-the-line without CRISPR.

  However, not every significant discovery is always thoroughly infallible. It is often challenging to deliver the CRISPR-Cas9 system to cells in necessary quantities without considering the clinical applications. Though scientists claim it to be 100% efficient and accurate, it still hasn't scaled up to such extreme heights. Applications like human germline editinga have raised serious ethical and societal objections10. They can be used to enhance desirable traits instead of curing diseases, which can be put to bad usage.11

  Certainly, such a revolutionary finding has improved the scope of research in ways that were impossible to venture into earlier. Just as the efficient-and-accurate CRISPR-Cas9 system removes the detrimental genes, CRISPR technology has made life a tad easy for scientists by significantly reducing the time duration for finding solutions to the existing problems. However, a huge scope still exists to know more about it. The technology is as inspirational as the words of Prof. Jennifer Doudna -"The more we know, the more we realize there is to know".12

Endote:
 a. Human germline editing: It is a bioengineering methodology by which the genome of an individual is edited in such a way that the change is inheritable. This can be achieved by genetically altering the germ cells, such as the egg and the sperm.


Bibliography

  1. Jinek M., Chylinski K., Fonfara I. et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity, SCIENCE 337 (6096), p. 816-821 (2012)
  2. Watson J., and Crick F. Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid, Nature 171, p. 737–738 (1953)
  3. Doudna J.A. and Charpentier E. The new frontier of genome engineering with CRISPR-Cas9, Science, 346 (6213) (2014)
  4. Jiang F. and Doudna J.A., CRISPR–Cas9 Structures and Mechanisms, Annu. Rev. Biophys. 46, p. 505-529 (2017)
  5. Deveau H., Barrangou R., Garneau J. E. et al. Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus, Journal of Bacteriology 190 (4), p. 1390-1400 (2008)
  6. Gootenberg J.S., Abudayyeh O.O., Lee JW. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356 (6336), p. 438-442 (2017)
  7. Ma, H., Marti-Gutierrez, N., Park, SW. et al. Correction of a pathogenic gene mutation in human embryos. Nature 548, 413–419 (2017).
  8. Sadhu, M.J., Bloom, J.S., Day, L. et al. Highly parallel genome variant engineering with CRISPR–Cas9. Nat Genet 50, 510–514 (2018).
  9. Wang, T., Zhang, H. and Zhu, H. CRISPR technology is revolutionizing the improvement of tomato and other fruit crops. Hortic Res 6, 77 (2019).
  10. Isasi R., Kleiderman E. and Knoppers KM. Editing policy to fit the genome? Science 351 (6271), p. 337-339 (2016)
  11. Human Genome Editing - Science, Ethics and Governance, The National Academic Press, Chapter 6, p. 137-162 (2017)
  12. Hayden EC. Human genome at ten: Life is complicated, Nature 464, p. 664-667 (2010)

Debjyoti Ghosh is currently a project student in the Department of Biological Sciences at IISER Kolkata. He is an enthusiast in synthetic biology (specifically protein engineering). He completed his BS-MS here in July 2020. Interestingly, football is his gravest addiction, and he seems to find time for his other interests besides his research.

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