(Absolutely disgusting laptop fan sounds, foreshadowing of the terrible audio quality to come)
Have you ever found yourself thinking,
"Gee, I wish I could alter targeted DNA at a nitrogenous base level..."?
Are the current gene editing models just not meeting your needs?
Are they lacking in precision and accuracy?
Lucky for you,
there's CRISPR Cas9
or,
Clustered Regularly Interspaced Short Palindromic Repeat,
a relatively (cough 2007) new gene editing tool that allows its user to make precise changes to the DNA of living cells!
CRISPR gene editing technology was developed from a CRISPR-associated protein-9 nuclease (Cas9) from
Streptococcus pyogenes,
which is a nasty little bugger, but that's besides the point.
The CRISPR Cas9 enzyme is utilized in bacterial cells as a
DEFENSE TACTIC
against foreign viral DNA (bacteriophages!)
Bacterial CRISPR identifies known viruses and inactivates their genes using priorly cut and copied viral DNA.
The pre-cut viral sequences are used as a guide for what area of the viral genome to slice up.
Once the genome of the virus is effectively ANNIHILATED,
the bacteria is safe from harm...
But how does CRISPR do this?
What mechanisms are at play, and what possible applications does it have in a clinical setting?
This diagram from the New England BioLabs demonstrates how viral DNA is targeted by the CRISPR Cas9 complex.
Before I begin, let me explain something quickly.
I will refer to Cas9 as both an enzyme and an endonuclease in the following explanations.
Although Cas9 IS an enzyme, its category AS an enzyme is an endonuclease.
This simply means that it cuts DNA apart at its sides rather than "unzipping" it down its middle.
So! With that said, let's begin.
(bumps my elbow on the table)
First, a segment of the foreign viral DNA, shown in this diagram as a green bar, is cleaved out of its original genome.
Then, the segment of DNA is copied into the CRISPR loci of the bacterial DNA sequence.
After this, through a process called CRISPR RNA
"bio-jin-ee-sis"
(Sound of disappointment)
(Amused) biogenesis,
CRISPR (RNA), or crRNA is effectively combined with tracrRNA to form guide RNA.
When a segment of foreign viral DNA is intercepted
This - Cas9 endonuclease uses the tracrRNA as a sort of handle,
while the crRNA guides the Cas9 enzyme to its matching viral sequence.
Once the sequence is located,
the Cas9-crRNA complex cleaves the matching foreign DNA.
Additionally, the PAM, or Protospacer Adjacent Motif, sequence
highlighted in red,
is a 2 - 6 base pair long region required for the cleavage of the viral DNA.
It indicates to the Cas9 that the region it is about to cleave is not bacterial DNA,
but instead harmful (DNA).
When Cas9 recognizes the PAM, it cleaves the DNA downstream.
Here's an animation of the crRNA matching with its corresponding viral DNA,
and Cas9 facilitating the double strand break!
So.
What do we know?
Well, we know that CRISPR can target
specific sequences of DNA
when given a target.
We also know that CRISPR's Cas9 endonuclease can facilitate a double strand break
at a specific part of a DNA sequence.
What can be done with this in experimentation on living, non-bacterial cells?
(I forgot how to speak English for the entire next part forgive me)
The approach to (the) CRISPR Cas9 system in a research setting
is... (???)
similar to the approach of a bacterial --
THAT a bacterial cell takes in ... purging i-itself of viral DNA.
To begin, researchers create a small piece of RNA attached to a
sh-shoort guide sequence
that binds to a distinct target sequence in the genome.
The guide and RNA complex --
(wrong "complex")
bind to a Cas9 enzyme
and the RNA itself is used to identify the desired section of DNA.
This region is then cleaved by the -- Cas9 guide and RNA complex.
After this,
the cell's own DNA repair mechanisms
are used by the researchers
to add, delete, or alter the genetic material.
So.... what can ... we do with this technology?
Animal model studies?
(Animal model studies flashes again)(laughs)
DEHvelopment of new medications?
Genetic modification of produce?
Let's take a look at what's already being done with CRISPR gene editing technology.
In 2016, Dr. Lu You, an oncologist from Chengdu, China,
used CRISPR to modify immune cells extracted from
lung (CANCER) patients blood to disable an oncogene.
He then injected these modified cells into the patient with the hopes that the cells will not multiply and cause metastasis.
Results from this trial have not been published.
CRISPR has also been used to eradicate HIV.
Researchers at the University of Pittsburgh and Temple University
have used CRISPR gene endngin
GENE EDITING technologies
to effectively inactivate HIV-1 in three different animal models.
CRISPR Cas9 is certainly exciting when considering new approaches to medicine, disease, and epidemic.
However, many questions are being raised about the ethics of gene editing,
as well as its implications.
New studies have also shown that CRISPR Cas9 has been producing unexpected mutations
in genes that were previously overlooked by researchers.
Although CRISPR shows a lot of promise,
it has a long way to go before it can be defivnitively used in practice.
