The essentials of a scientists gene editing toolbox: CRISPR cas9 system

In my previous post I gave a brief introduction into the CRISPR system. Which effectively is like an immune system adopted by bacteria to protect themselves against viral infections. But still the question is....

How are scientists using this to edit genes in research?

Sit back and relax and for the next part of the CRISPR story to unfold....(crisps not necessary)

Professor Jennifer Doudna and  Emmanuelle Charpentier were studying the bacteria Streptococcus Pyogenes  and discovered the cas9 system (note cas= CRISPR associated proteins). Streptococcus Pyogenes  only have cas9 type cas proteins.

Cas9 Protein (blue) in action with gRNA (cyan) and DNA (magenta)

So, what is the BIG deal about this cas9 protein?

Cas9 protein has a  nuclease region in its major structure-i.e it has an areas within the protein that can cut DNA.  Streptococcus Pyogenes makes two long strips of ribonucleic acid (RNA). RNA is similar to DNA, it is made up of nucloetide bases but unlike DNA RNA is single stranded and contains the nucleobase uracil instead of thymine.

Comparison of DNA and RNA

Cas9 holds both CRISPR RNA (crRNA) which contains a spacer segment which matches up to the corresponding viral RNA. CrRNA is effectively a copy of part of the CRISPR genes (DNA). In my previous post I covered a bit about the spacer segments in the CRISPR system matching up to viral DNA. Cas9 also holds trcrRNA which holds the crRNA in place in the cas9 protein.This allows the viral DNA to be cut by the nuclease activity of the cas9 protein. As the crRNA matches up the viral DNA to be degraded and disposed of and the trcrRNA anchors the crRNA in place. 

Cas9 CRISPR system with viral DNA matching up with crRNA and CRISPR spacer

Scientists exploited the cas9 system by modifying the crRNA by putting their own RNA sequence in to replace the spacer segment and connecting the crRNA and trcrRNA together to form a crRNA-trcrRNA chimera. A chimera is a mythological creature which is made up of different parts of different animals. Similarly in molecular genetics a chimera is a DNA or RNA molecule formed from two or more organisms by laboratory manipulation. This chimera in the cas9 CRISPR system is often referred to the guide RNA or gRNA. 

gRNA and cas9

In short, the cas9  CRISPR system has the cas9 protein which cuts the DNA and the gRNA which guides where the DNA is going to be cut.

An exciting tool for gene editing!

Steps to gene editing using the cas9 CRISPR system....

1) Find and identify the region of DNA that you want to cut

2) Create a guide RNA or gRNA that has a corresponding piece of RNA to the DNA that you want to cut

3) The DNA will be fed through the cas9 CRISPR system and the cas9 protein will cut the selected DNA sequence and inactivate that gene.

Of course the cell will naturally try and repair this break in the DNA by a number of mechanisms causing a mutation. But essentially the selected gene has been inactivated.

Further  from inactivating the gene, a new gene can be inserted.

4) Inserting a host piece of DNA into the cell along with the cas9 CRISPR system so that when the DNA is cut this piece of DNA bridges the cut and the cell repairs and incorporates this new gene into the DNA.

Tada! Genes edited.

Next time...

 In my next post I'll be looking at some of the applications of the cas9 CRISPR system and exploring its future implications. Where will this gene editing take us?

CRISPR, not a new savoury snack but an exciting tool for scientists-an introduction

An introduction to CRISPR in a short series on gene editing

CRISPR is a biological system scientists are exploiting using to edit genes in research. 

I  hear you  saying why, what, where and HOW??? Well grab some CRISPs and hold on tight for this whistle stop tour of CRISPR system!

Firstly, a gene is a short sequence of DNA- deoxyribonucleic acid. Hailed for being the blueprint or the code for life DNA is made up of two strands of four different nucleotides, adenine (A), cytosine (C), guanine (G) and thymine (T) which form a code for the production of proteins in the cell. Every three nucleotides (beads) on one strand form a code for an amino acid.

DNA made up of four nucleotides

In  some diseases there can be changes in this code of nucleotides, for example in cystic fibrosis there is a deletion of three nucleotides which form the code for the the 508th amino acid (phenylalanine) in a protein which lies across the membrane (enclosing) of cells. This is the molecular basis of cystic fibrosis and shows how genetic changes can have a huge impact on cell function. Hence being able to edit this genetic code could have huge implications on health and disease. To explore more about how the amino acid sequence affects proteins have a read of my previous blog post on protein folding and organisation.

What if we could change these errors in the code? How can we ensure that the DNA (which is so long) is edited in exactly the right place? What can we use to do this?

In comes CRISPR (Clustered regulatory inserted short pallindromic repeats-SUCH A mouthful) which was  co-discovered in 2012 by Professor Jennifer Doudna and  Emmanuelle Charpentier who were studying how prokaryotes (i.e bacteria etc) defend themselves against viruses and virus related microorganisms such as phages. Yes, bacteria does have its own form of immune system.

When a bacterium is attacked by a virus, the virus inserts its own genetic material which is then replicated by the bacterium and is used to make new viruses.
When a bacterium has a CRISPR system, it has sequences of identical DNA which are evenly spaced by DNA which is has a unique sequence. This spacer DNA is important as it matches up with viral DNA. 

This CRISPR system is associated with a set of genes called Cas (CRISPR assoicated genes) which encode for proteins that unwind and cut the DNA When a bacterium is infected by a virus and the viral DNA is inserted and the spacer DNA, so the CRISPR gene that matches the inserted viral DNA is copied into a transcript called CRISPR ribonucleic acid (crRNA). The crRNA fits into the Cas protein and this breaks up the viral DNA preventing the infection.

CRISPR system when viral DNA matches CRISPR spacer

When viral DNA is inserted into a bacterium that does not have a matching CRISPR spacer, a different kind of Cas protein is produced that copies this viral DNA to create a CRISPR spacer for the next time it infects as well as breaking down the viral DNA.

CRISPR system when viral DNA does not match CRISPR spacer

That is is the CRISPR system in a crisp packet! Scientists Professor Jennifer Doudna and  Emmanuelle Charpentier studies the CRISPR system in bacteria Streptococcus Pyogenes. This enabled them to develop a tool for gene editing.

Now you know a little bit more about the basic CRISPR system, sit tight for part 2 to explore how this system is used for gene editing and to think about the future implications.