CRISPR

Overview.

Background.

Technique.

Potential applications.

Overview.

The Human genome was sequenced, in the beginning of the 21st century.

Scientists have identified and sequenced, the genome of many species.

They are now in the process of identifying the role of a gene, or a system of genes,

in the metabolic process of life.

Recently using a technique, called CRISPR,

scientists have succeeded in editing genes.

By doing this, we can modify the basic processes of life.

We could potentially cure or eliminate diseases.

This technique has already been tried, on insects and animals.

It can be potentially used in human beings also.

This requires a more thorough understanding of the genetic functioning.

We need to build some more safeguards, before using this technique,

more widely in human beings.

One interesting and powerful application, of gene editing, is related to malaria.

Some species of mosquitoes, transmit the disease from one human being to another.

This is capable of spreading the disease, throughout the population.

Malaria is widespread in many countries.

Scientists using CRISPR, have been able to modify the genes,

of the mosquito, so that it is incapable of carrying and transmitting the disease.

Using another powerful technique called gene drive, it is possible to spread,

this genetically modified trait, to an entire population of mosquitoes.

This offers the possibility of eliminating malaria, in many countries.

CRISPR stands for Clustered Regularly Interspaced Short Paiindromic Repeats.

Background.

The technique of CRISPR, was learnt by observing the way,

bacteria dealt with invading viruses.

Bacteria viruses injects its DNA material to host cells.

The genome of the bacteria, has DNA repeats,

with spacers are unique sequences in-between.

These spacers are derived from DNA of viruses,

that prey on bacteria.

Each spacer gene, has the DNA sequence of a virus,

that previously infected the bacteria.

This region in the genome, is a DNA library of all enemy viruses.

It provides the sequence, to identify and destroy an invading virus.

The spacer DNA is transcribed, as RNA, and forms a complex with a protein called Cas.

Cas stands for CRISPR Associated System.

Cas is a endonuclease which cuts DNA.

Cas genes makes Cas proteins.

The RNA/Protein complexes drift through the cell,

looking for viral DNA.

When it finds it, it latches on to it, base pairs with it,

and cuts the viral DNA, effectively disabling the virus.

If it is a new virus, the adaptive immune system of the bacterium,

takes up the genetic material of the virus,

and inserts it into its own DNA.

Next time the virus attacks the bacteria, it has a ready gene sequence,

to generate the RNA/Cas complex to fight and eliminate the virus.

There are 3 main stages in the CRISPR technique.

Adaptation.

Expression.

Interference.

Adaptation:

This is the stage, where the bacteria incorporates, or implants,

the viral genome, into its own genome.

The DNA of the virus, that tried to attack the bacterium,

is found between the short DNA repeats, called spacers.

During adaption these new spacers, are inserted into the CRISPR locus.

Expression:

In this stage the inserted genes are expressed.

CRISPR is transcribed into CRISPR RNA.

Interference:

This is the stage of detection and degradation, of mobile genetic elements,

by CRISPR RNA and Cas protein.

This cuts the invading virus DNA,

and protects the bacteria from infection.

Scientist borrowed this concept from nature.

They designed their own RNA and similar system called CRISPR/Cas.

This can target any DNA, in any location, in any organism.

Technique.

CRISPR comprises of a CRISPR RNA,

which corresponds to the target DNA.

It also has a tracer RNA, which holds the CRISPR RNA in place.

The combination of CRISPR DNA and tracer RNA,

is called guide RNA.

The commonly used Cas protein, is the Cas9 protein.

The guide RNA, and the Cas9 protein, together is called the CRISPR complex.

The guide RNA, is a short synthetic RNA.

It is composed of a scaffold sequence for Cas9 binding.

It has a user defined "spacer" or targeting sequence.

This sequence could be typically about 20 nucleotides.

We can change the genomic target of Cas9,

by changing the sequence in the guide RNA.

This system is used to transfect living cells.

This happens within the nucleus of the cell.

The Cas9 protein will unzip the DNA and match it to the target.

If it matches, it will use tiny molecular scissors, to cut the DNA.

After Cas9 cuts the DNA, the cell would try to repair the break,

using the copy of the gene.

This is an error prone process, and could alter the gene sequence.

This can disable a particular gene.

CRISPR can modify both copies of the gene.

This makes it very efficient, ensuring that the target gene is disabled.

This method is useful for disabling a gene.

For example, to disable a gene associated with the disease.

It can also target many genes associated with the disease.

Another interesting feature of CRISPR,

is the capability to replace a target gene, with a desired gene.

For example, a defective gene can be replaced with a good gene.

To do this, a desired DNA sequence, is provided along with the CRISPR/Cas9 system.

It is used by the cell's repair pathway,

as a template to reconstruct the disrupted gene sequence.

This kind of alternations to genes is permanent.

CRISPR techniques are getting better and better.

We can selectively turn on and off, target genes,

and fine tune their expressions, without altering their gene sequence.

In other cases we can target a specific gene, and replace it with a desired gene.

We can also insert a desired gene, at a specific location.

CRISPR can target many genes at once.

This is of great advantage, when working with diseases,

which involves many genes.

CRISPR can be applied to somatic cells, of a tissue or an organ.

Stem cells are mostly uncommitted cells, which can differentiate itself,

into specific cells.

For example, stem cells are present in the bone marrow.

These are called haematopoietic cells.

These cells can generate any type of the many blood cells that we have.

CRISPR can be applied to stem cells.

In this case, the change will propagate to all types of blood,

that is produced from the stem cell.

Germ line cells, are involved in reproduction.

In this case, change introduced will be passed on to all future generations.

Potential applications.

CRISPR can be used for disease resistance.

It can be injected into developing embryos.

It can be done on a fertilised egg,

allowing creation of transgenic organisms,

with targeted mutations.

It can bring back extinct species.

It can introduce deleterious genes into malaria carrying mosquitoes,

effectively disabling them from carrying and transmitting their disease.

It can modify animal organisms, to produce organs,

which can be transplanted to humans.

It is also being used to breed custom pets like fish and dogs.

One of the most powerful use of these techniques,

is to alter germ line cells, which are involved in reproduction.

This enables altering of genetic heritage of future generations.