The foundations of Genetic Editing: CRISPR 

This article covers CRISPR, a genetic editing technique, as such it will assume some basic knowledge of genetic editing. If you are unfamiliar with the process, I highly recommend reading The Basics: Genetic Editing first. 

There are many different techniques of genetically engineering cells. One such technique is CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats. This was originally discovered as a feature of the bacterial genome and forms part of the bacterial defence system but has been successfully adapted into a technique for genetic editing.  

CRISPR as bacterial defence. 

This genomic CRISPR array is used to target invasive viruses, it acts as a memory bank that stores DNA fragments of previous invaders. In response to infection the CRISPR array is transcribed into RNA and the fragments separated. Each fragment binds to a Cas protein (CIRSPR associated protein) which targets the CRISPR/Cas complex towards any complementary DNA of the invading virus.   

The Cas protein has nucleolytic activity, which means when it is activated by the binding of the RNA CRISPR fragment, it cuts through the backbone of the invasive DNA. Therefore, any sites on the invasive DNA with complementary DNA to the RNA CRISPR fragment are broken preventing the virus from hijacking the cell. The viral DNA fragments are then incorporated into the CRISPR array strengthening the cell’s response to subsequent invasions.  

CRISPR/Cas in gene editing 

Genetic engineering can be achieved through the manipulation of this simple system. The major components of the system are the Cas protein and the RNAs that activate and target the complex to the DNA. Through combining the RNA elements in one gRNA (guide RNA) the process is simplified even further. This means that in order to target the DNA only two components are needed to be supplied into the cell.  

Once in the cell the gRNA targets the Cas protein to a specific location in the genome, creating strand breaks. This is the point at which the DNA can be changed. The cell has several mechanisms for repairing these breaks in the DNA. The first trims the bases either side of the break then glues the end back together. This is a good method to use if the desired modification is to disable the functioning of the gene. Whereas in the other method of repair requires a template which is then copied into the DNA ensuring that no information is lost. This template must match the ends either side of the break to trigger this cellular repair response however this response can be manipulated through supplying a template alongside the gRNA and Cas protein. The supplied template can contain a new gene thereby facilitating the integration of a novel gene into the genome. 

There are many considerations for using the system including; whether the aim to silence or add in a new gene, if there are any other sites where the gRNA might bind (off-target effects) and the Cas protein used. There are many different Cas proteins that can be used either originating from different organisms or mutated to for a specific purpose although Cas9 is the most commonly used. For this reason, the CRISPR system can also be written as CRISPR/Cas or CRISPR/Cas9. 

Summary

The CRISPR/Cas system was found in the genome of bacteria as a defence system but has been harnessed for genetic editing.  As a system for genetic engineering it can target a specific location on in the genome and create a double strand break. This accurate break allows for the cellular repair mechanism to introduce the desired changes to the DNA.  

Further Reading 

Nature Video on Youtube – CRISPR: Gene editing and beyond  

A visual journey through the methods behind CRISPR.

New Scientist – What is CRISPR? 

A good place to start with the New Scientist’s contribution for understanding CRISPR.  

Relevant Articles

A review article covering the role CRISPR plays in bacterial defence.  

Fineran, P. C., & Charpentier, E. (2012). Memory of viral infections by CRISPR-Cas adaptive immune systems: Acquisition of new information. Virology, 434(2), 202–209. https://doi.org/10.1016/j.virol.2012.10.003

A review article covering CRISPR in relation to genetic editing.  

Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262–1278. https://doi.org/10.1016/j.cell.2014.05.010

Learn More from Talking Biology

The Basics Series

DNA

A look at how the DNA molecule and how that relates to genes. 

Genetic Editing

 An overview of what is meant by genetic editing, how it is done and the reasons behind it.