CRISPR Cas-9 technology

 

The CRISPR-Cas 9 is a breakthrough in genetic engineering that enables geneticists and medical researchers to edit parts of the genome by adding, removing or altering sections of the DNA sequence.

This technique has been developed by Jennifer Doudna and Emmanuelle Charpentier that earned the two researchers the Nobel Prize in Chemistry in 2020.

The CRISPR-Cas 9 technology is highly esteemed in the biotechnology and medical field as it enables genome editing in vitro highly precisely.

Why the name CRISPR-Cas 9?

CRISPR (clustered regularly interspaced short palindromic repeats) - CRISPR is the family of the DNA sequences used in this technology. These DNA sequences are extracted from prokaryotes.

Cas-9 - It is a a dual RNA-guided DNA endonuclease enzyme. This enzyme has a significant role in the immunological defense system of certain prokaryotes against viruses and plasmids.

Structure of CRISPR

Applications of CRISPR

1.   Gene editing

CRISPR technology has been applied in the food and farming industries to engineer probiotic cultures and to immunize industrial cultures (for yogurt, for instance) against infections. It is also being used in crops to enhance yield, drought tolerance and nutritional value. 

2.     Diagnostic tool

CRISPR associated nucleases have shown to be useful as a tool for molecular testing due to their ability to specifically target nucleic acid sequences in a high background of non-target sequences.

Cas 9 structure

Fig 1. Cas 9 (orange and red molecule) acting on DNA (blue molecule)

Major Components of the CRISPR Cas-9

The CRISPR-Cas 9 genome editing is performed with a Type II CRISPR system and the major components involved in this system are as follows:

Component	Function
crRNA	Contains the guide RNA that locates the correct segment of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form), forming an active complex.
tracrRNA	Binds to crRNA and forms an active complex.
sgRNA	Single-guide RNAs are a combined RNA consisting of a tracrRNA and at least one crRNA.
Cas9	An enzyme whose active form is able to modify DNA. Many variants exist with different functions (i.e. single-strand nicking, double-strand breaking, DNA binding) due to each enzyme's DNA site recognition function.
Repair template	DNA molecule used as a template in the host cell's DNA repair process, allowing insertion of a specific DNA sequence into the host segment broken by Cas9.

How does the CRISPR-Cas 9 work

The CRISPR Cas 9 system relies on a ''DNA repair machinery'' mechanism where two main molecules are involved in introducing a change (mutation) into the DNA namely the 

• Cas 9 enzyme 

• The gRNA (consisting of a small piece of pre-designed RNA sequence located within a longer RNA scaffold).

   

First let us understand what leads to the production of the Cas 9 molecule in bacteria. 

• When a bacterium detects presence of the viral DNA, it produces 2 types of small RNA molecules one of which that contains the sequence that matches the invading virus.
  

• These 2 RNA forms a complex with the Cas 9 enzyme. 
  

• The scaffold parts of the gRNA binds to the target DNA and the pre-designed sequence guides Cas 9 to the right path of the genome.
  
  
  
• Cas 9 follows the gRNA to the same location in the target DNA sequence and makes cut across both DNA strands.

                                             

• At this stage, the cell recognizes that the DNA is damaged and attempts to repair it.   
• This DNA repair machinery is what allows scientists to cut, add or remove pieces of DNA.

                                                 

Potential of CRISPR-Cas 9 

• The CRISPR-Cas9 system can be used to manipulate and excise stretches of DNA that have profound relevance for human health, ranging from aggressive malignancies to mitochondrial storage diseases, as well as potential applications in the prevention and treatment of infectious diseases such as human immunodeficiency virus, influenza virus, and malaria, among many others.

• Scientists are showing interest for its potential use in editing germline.

Application of the CRISPR Cas-9 

• Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time.

• It has since been used in a wide range of organisms, including baker's yeast (Saccharomyces cerevisiae),  the opportunistic pathogen Candida albicans, zebrafish (Danio rerio), fruit flies (Drosophila melanogaster), ants (Harpegnathos saltator and Ooceraea biroi), mosquitoes (Aedes aegypti), nematodes (Caenorhabditis elegans),plants, mice, monkeys and human embryos.

Video resources

References

•         Anon (n.d.) What is CRISPR-Cas9? [Online]. yourgenome. Available from: /facts/what-is-crispr-cas9 [Accessed: 7 June 2021].
•         Anon (2021) CRISPR. [Online]. 6 June 2021. Wikipedia. Available from: https://en.wikipedia.org/w/index.php?title=CRISPR&oldid=1027145116 [Accessed: 7 June 2021].
•         McCarthy, M.W. (2020) Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine. Expert Review of Anti-Infective Therapy. [Online] 1–7. Available from: doi:10.1080/14787210.2020.1761333.