What is CRISPR/Cas9 gene editing?
CRISPR/Cas9 is a
gene-editing tool used to modify, delete, or correct specific regions of DNA.
It is faster, cheaper,
and more accurate than previous techniques of editing DNA, and it has a wide
range of potential applications.
CRISPR/Cas9 stands
for?
CRISPER Stands for Clustered
Regularly Interspaced Short Palindromic Repeats: These are short repetitive
palindromic DNA sequences present in a genome
Cas 9: Cas9 is a
CRISPR-associated (Cas) endonuclease, that cut DNA at a location specified by a
guide RNA.
The system was
originally discovered in bacteria as a defense mechanism against viral attacks.
What is guide RNA?
A guide RNA (gRNA) is
a short piece of RNA complementary to the target DNA sequence used to guide the
Cas9 enzyme to a specific DNA sequence.
The Cas9 enzyme then
cuts the DNA at that location, which allows researchers to add, remove, or
alter the DNA sequence.
How CRISPR/Cas9 gene
editing works?
CRISPR/Cas9 is used to
treat a patient with sickle cell disease caused by a specific DNA mutation.
5 Steps in CRISPR/Cas9
gene editing
Step 1: Selection and
preparation of cells
The patient's hematopoietic
stem cells from the bone marrow is isolated and cultured.
Step 2: Synthesis of
gRNA complementary to the target sequence
Scientists knew exactly
the mutation and normal gene sequence of hemoglobin gene. So, guide RNA is
synthesized that is complementary to the target DNA sequence that is to be
edited.
Step 3: Formation of
Cas 9 guide RNA complex and transfection to the target cells
Cas 9 enzyme binds to
guide RNA by base pairing interaction forming a complex and that is transfected
to the target cell by various methods such as microinjection, electroporation
etc.
Step 4: DNA cleavage
and DNA repair
Cas 9 guide RNA
complex binds to the complementary target sequence and cuts
the DNA at the target location making double-stranded break (DSB).
The cell's DNA repair
machinery then repairs the DNA at the cut site. There are two main ways that
the DNA can be repaired:
Non-homologous end
joining (NHEJ): This is a quick and efficient way to repair the DNA, but it
often results in small insertions or deletions in the DNA sequence.
Homologous
recombination (HR): This is a more precise way to repair the DNA, but it is
also slower and less efficient.
Step 5: Infusion of edited
cells back to the patient
The edited stem cells
are then infused back into the patient. The edited stem cells can develop into
new healthy red blood cells that do not have the sickle cell mutation.
This approach is still
in the early stages of development, but it has the potential to be a promising
treatment for sickle cell disease.
Check your understanding: Multiple choice on CRISPR/Cas9 Technology.