CRISPR is the newly discovered REVOLUTIONARY
tool that would allow scientists to change
AT WILL any DNA sequence of, presumably, any
living organism in a precise manner.
Unlike any other previously developed techniques
of gene editing, CRISPR is REMARKABLY simpler,
faster and cheaper.
CRISPR is part of a naturally occurring defense
mechanism found in many bacteria.
The bacteria use CRISPR to SPECIFICALLY snip
the DNA of invading viruses.
CRISPR stands for “Clustered Regularly Interspaced
Short Palindromic Repeats” - a region of
bacterial genome that contains short DNA repeats
with unique sequences, or spacers, in between.
These spacers are derived from DNA of viruses
that prey on the bacteria.
The CRISPR region is essentially a DNA library
of all enemies that need to be RECOGNIZED
and destroyed.
After being transcribed, individual pieces
of spacer RNAs form complexes with a protein
named Cas, for CRISPR-ASsociated protein.
Cas is an endonuclease – an enzyme that
cuts DNA.
These RNA/protein complexes then drift through
the cell, looking for matching viral DNA.
If a match is encountered, the RNA latches
on, base-paring with it; Cas protein then
cuts the viral DNA, disabling the virus.
Scientists have isolated this system, and
by designing their own spacer-RNAs, they can,
in theory, target any DNA sequences in any
organism.
The system has indeed worked in all organisms
tested so far.
The current CRISPR system consists of two
components: a guide RNA and a Cas protein
named Cas9.
The guide RNA is a short synthetic RNA composed
of a “scaffold” sequence necessary for
Cas9-binding and a user-defined “spacer”,
or “targeting” sequence of about 20 nucleotides
long.
One can change the genomic target of Cas9
by simply changing the targeting sequence
present in the guide RNA.
The entire system is designed in a plasmid
that is subsequently used to transfect living
cells.
Some applications of the CRISPR system include:
- Disabling, or knock-out, a particular gene:
After Cas-9 cuts the DNA, the cell would try
to repair the break.
The more efficient repair pathway in the cell
is ERROR-PRONE and would most likely result
in a loss-of-function mutation in the gene
of interest.
As CRISPR modifies BOTH copies of the gene
at the same time, generation of knock-out
animals and cell lines for gene function studies
has never been more efficient.
Moreover, MULTIPLE genes can be targeted in
one manipulation, making this technique an
extraordinarily powerful tool for studying
complex genetic traits or diseases that involve
many genes, such as cancers.
- Introducing precise modifications to the
target DNA: If a desired DNA sequence is provided
together with the CRISPR/Cas-9 system, it
can be used by ANOTHER repair pathway as a
TEMPLATE to reconstruct the disrupted gene
sequence.
The desired changes stay permanently and are
also transmitted to future generations.
This can be used, for example, to swap a mutated
copy of a gene with the good version, thereby
restoring the gene’s function.
- Modifications to the Cas9 enzyme have extended
the application of CRISPR to selectively turn
ON and OFF target genes, fine-tune their expression
WITHOUT permanently altering the gene sequence.
Since its discovery, CRISPR technology has
been used extensively in animal research to
engineer disease-resistant livestock; bring
back extinct species; introduce deleterious
genes into malaria-carrying mosquitoes; and
modify pig genome to make pig’s organs suitable
for transplant into human.
Due to its relative simplicity, CRISPR has
also been employed to create “custom designed”
pets such as mini-pigs with customized coat
patterns, colorful koi fish and dogs with
certain desirable traits.
The CRISPR zoo is growing rapidly but so are
the ethical concerns and fears of possible
ecological disasters.
In human, while CRISPR is proven to be a powerful
tool to study various diseases, it is deemed
NOT YET ready for clinical applications.
This is because the Cas enzyme occasionally
still cuts in the wrong place and hence cannot
be used to introduce permanent changes to
people.
Modification of human germlines to alter genetic
heritage of future generations may also lead
to unwanted far-reaching consequences and
is prohibited by most countries.
