written by: Sarah Ning
graphics by: Jaimini Patel
Introduction
Imagine if there was a way for you to genetically edit parts of yourself, whether that means eliminating hereditary cancer, or choosing desirable traits for your children. Have you ever wondered if there were a way to bring back extinct species like the woolly mammoth from the dead? Or if there were a way to invent hypoallergenic food, or even to get rid of allergies completely? In recent years, a revolutionary scientific discovery emerged, which can make all those “what ifs” a reality! Recognized as one of the most significant scientific breakthroughs of the century, CRISPR is a gene-editing tool that enables geneticists and researchers to edit portions of the genome by removing, adding or changing sections in the DNA sequence. The major breakthrough of the CRISPR technology came in 2012 when research teams in the United States and Europe—led by 2020 Nobel Prize winners Jennifer Doudna and Emmanuelle Charpentier—demonstrated how this technology acts as a ‘cut and paste’ tool for editing a genome. In short, CRISPR is currently the most precise, versatile, cheap, and fast option for genetic manipulation.
But what exactly is CRISPR? CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, referring to the unique organization of short, palindromic repeated DNA sequences found in the genomes of certain microorganisms, such as bacteria. A CRISPR locus is a certain area in the genome where a particular DNA sequence is repeated over and over again, with sequences of spacers in between. Next to these sequences are Cas proteins, the most well known and most commonly used being the Cas9 protein. Now that you’re a bit more familiar with the topic, let’s delve deeper into the history and methods behind CRISPR.
The History behind CRISPR
The idea of CRISPR technology was introduced in 1987 after Japanese scientist Yoshizumi Ishino studied how the Escherichia coli bacteria fought viral infections. The results from this experiment showed that E. coli had an adaptive immune system, known as CRISPR, that kept them protected from bacteriophages. Many bacteria and archaea evolved their CRISPR system as a natural defence mechanism against pathogens, acquiring adaptive immunity. Since then, the CRISPR-Cas system has been found in 50% of bacterial genomes and 90% of archaeal genomes. So, how exactly does this built-in gene-editing system work in these microorganisms to combat against pathogens? This can be broken down into 5 steps:
When a virus attacks the bacterium, the CRISPR system snips out sections of the viral DNA known as protospacers. These protospacers are then integrated into the bacterium’s CRISPR locus as new spacers.
If the same virus invades again, the CRISPR-Cas system will match the virus’ protospacer sequence to the identical spacer sequence in the bacterium’s genome.
The bacterium will transcribe these spacer sequences to RNA. This RNA chain is then cut into shorter pieces called CRISPR RNA (crRNA).
The corresponding crRNA acts as a guide for the Cas protein and will recognize a short 2-6 base pair sequence in the virus’ genome called a protospacer adjacent motif (PAM), which is found near the target gene.
The Cas protein will cleave the viral sequences. The virus is destroyed and does not infect the bacterium.
How does CRISPR work as a gene editing tool?
Scientists have altered the natural prokaryotic CRISPR system to be used as a modern gene-editing technology. This CRISPR technology creates precise changes in the genomes of various organisms, including humans, who do not have a built-in CRISPR system.
There are two key molecules involved: the Cas9 protein complex and the guide RNA (gRNA):
Cas9: An enzyme that acts as ‘molecular scissors’ to cut the two strands of DNA at a specific site in the genome, in order for the DNA to be edited.
gRNA: A small piece of pre-designed RNA sequence (~20 bases long) that recognizes a particular DNA sequence and will guide the Cas9 protein to the location in the genome. This acts as the crRNA that is found in bacterial and archaeal cells.
So how does the process work?
Decide which genome sequence to modify.
Design the gRNA sequence that will have complementary base pairs to the DNA sequence being targeted.
The gRNA is attached to the Cas9 protein to form a complex. The Cas9 complex is introduced to the target cells.
The complex locates the PAM and the Cas9 protein cuts the DNA.
The double-stranded breaks in the DNA are repaired by one of two cell repair pathways:
Non-homologous end joining:
The host cell has its own DNA repair mechanism that is error-prone and often introduces frameshift mutations that disable the function of the gene.
Homology directed repair
A precise/accurate process of replacing a mutant gene with a healthy copy using specific deletions or insertions of genes.
Now that we have a better understanding of how this gene editing technology works, it is also fascinating to learn about all the different applications of CRISPR. Stay tuned for next week’s blog post on all the ways that CRISPR can be used!
Glossary
Palindromic: sequence made up of nucleic acids within a double helix of DNA or RNA that reads the same from 5' to 3' on one strand and 3' to 5' on the complementary strand.
Spacers: regions of non-coding DNA between genes.
Bacteriophage: virus that infects and replicates within bacteria and archaea.
Pathogen: broad term for a biological agent that causes disease or illness to its host.
Archaea: unicellular prokaryotes typically found in extreme environmental conditions
DNA: Stands for deoxyribonucleic acid. Carries an organism’s unique genetic code.
RNA: Stands for ribonucleic acid. Acts as a messenger carrying instructions from DNA for controlling the synthesis of proteins
Adaptive immunity: acquired immune system that occurs after being exposed to an antigen (ie. from a pathogen or vaccination)
References
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