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?