You may have heard of a technique now commonly being used in plant science: CRISPR/Cas9. But what is this? and why is it causing such a buzz in the science world?
What is CRISPR/Cas9?
CRISPR/Cas9 is a new(ish) tool used for targeted genome editing. This means that a specific change can be made to the DNA of an organism- this can be via deletion, insertion or replacements .
Genome editing in more detail:
For genome editing of plants, engineered nucleases are used- these basically act as scissors which are able to cut in certain locations of the genome. This cut makes a site specific double strand break: both strands of the DNA helix are cut. The induced break is then repaired by one of two processes: non-homologous end joining, or homologous recombination. The method of DNA repair isn’t 100% accurate, resulting in targeted mutations.
Genetic engineering allows us to study the gene functions within organisms, and within plants can lead to the production of plants with useful biological traits.
Before CRISPR/Cas9, 3 families of engineered nucleases were commonly used for genetic engineering: Meganucleases, Zinc-finger nucleases and transcription activator-like effector based nucleases (TALENS). However, although effective, these endonucleases have some drawbacks:
- Expense- you need to make your nuclease specific for the DNA sequence you want to target, and this can be pretty costly.
- Efficiency- you may have several targets in a DNA sequence for your nuclease, and may lead to off-target mutations leading to toxic changes to the genome.
- Speed- construction of sequence-specific enzymes can be time consuming.
What makes CRIPSR/Cas9 better?
- It is very versatile
- It is very precise
- It has a pretty good efficiency
- It is less time consuming
- It’s relatively cheap
- There are a lot of potential applications for CRISPR/Cas9…
More about CRISPR/Cas 9
CRISPR stands for: Clustered regularly interspaced short palindromic repeat.
Originally the CRIPSR/RNA system was used in bacteria, and only got converted to a gene editing tool in 2012- but since then, it has blown up the science world!
The CRISPR/Cas9 system is made from two key molecules:
An enzyme= Cas9, which acts as the scissors to cut the DNA
Guide RNA= a piece of RNA which is a bit of pre-designed RNA sequence, about 20 bases long, which recognises the DNA sequence you are targeting. The guide RNA guides the Cas9 enzyme to the location where it is meant to cut the DNA, this is what makes CRIPSR/Cas9 so precise!
When the enzyme cuts the DNA, the cell recognises the damaged DNA and sets out to repair it. We can use the DNA repair machinery at this point so that changes can be introduced to the genome- whether that is an introduction, deletion or alteration to the genome. The changes to the genome can be small- with just a single base pair changed, or can involve the introduction or removal of a entire gene.
With its high efficiency, and relative simplicity, CRISPR/Cas9 can be used in many applications from gene mutation, activation and repression, through to addition or replacement of genes. This, plus the fact that it produces much less off-target events has made it a tool which no one can get enough of!
A big benefit:
One of the main benefits of CRISPR/Cas9 is that the genome of a plant can be selectively edited without relying on agro infiltration, or viral infection (as with stable transformation techniques). This means that no external DNA is added to the target plants genome- therefore meaning that the generated plant is not GMO.
This is a particular benefit to agricultural science- beneficial gene modifications can be made to crop plants much faster, without the need for selective crossings and back crossings which can take many, many years, all without the need for GMO plants. The use of CRISPR/Cas9 in plant science is extremely positive, and is expected to be applied to many different plant-based projects in the future.
Are there any problems with CRISPR/Cas9?
Targeting: the guide RNA is made of 19-20 bases, and are complimentary to the target sequence. However, not all the base pairs need to be a match for the guide RNA to bind. This means that the guide RNA will sometimes bind to the target location, as well as the incorrect location, leading to a mutation in the wrong place.
This is something that many scientists are currently looking into- so that in the future we can get even more precise and efficient gene editing.
Related Blog posts:
Sources and further reading:
Overview article about genome editing: https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting
(Research paper, may not be available to all) http://science.sciencemag.org/content/346/6213/1258096
(Research paper, may not be available to all)