Making Gene Therapy CRISPR

By Alyssa Evangelista

CRISPR-Cas9 is a groundbreaking technology that can alter the genome for the better or
for the worst. For a long time now, we have known genetic mutations that underlie disease,
says Feng Zhang in a video by Stat News. ...if we know what mutations cause disease, why not
just go in and replace it and revert it back to the normal sequence?
CRISPR does just that. It has the power to change the coding of DNA, the nucleic acids
that provide the blueprint for life. Yet, researchers are scratching their heads and debating about
the issues it presents.

What is CRISPR?

Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR-Cas9 for short,

is a newly discovered gene editing system that was originally found in bacteria by scientists at
the University of California Berkeley. They observed that this system served as the bacterias
immune defense because it remembers and copies a virus DNA and cuts it out the next time the
same virus tried to infect the bacteria. This was quite an interesting find. Then the scientists
thought, How can we apply this to other organisms? Thus, the gene revolution began.
As a gene editing tool, CRISPR utilizes an enzyme, called Cas9, to cleave or cut the
DNA at the target gene. A guide RNA (gRNA) steers Cas9 to this gene to ensure that it reaches
its target. From there, the cell will repair the cut with a mutation, or researchers can choose to
insert another gene.
This new technology has huge potential. Theres talk about how it can be used to wipe
out mosquito populations that carry Zika. It can eliminate deadly diseases such as AIDS and
cancer and prevent genetic diseases before a baby is even born. It could even bring extinct
species back, like in the movie Jurassic Park.
CRISPR may be the answer to many of our problems. However, there are many safety
concerns that make this system is so controversial. One of these concerns is off-target activity.

Missing the Bullseye

CRISPRs got an off-target problem; it doesnt always reach its intended gene. Why?
Here are two reasons.
First, all of the bases of the gRNA dont have to match up to the target gene in the DNA.
In most cases the guide RNA consists of a specific sequence of 20 bases. These are
complementary to the target sequence in the gene to be edited, says YourGenome, a website
produced by the Public Engagement Team and the Wellcome Genome Campus in the UK.
However, not all 20 bases need to match for the guide RNA to be able to bind.
 Sometimes only 18 out of 20 bases need to match in order to bind to a gene. You may
think that this isnt really a huge deal, but one mismatched base can lead to Cas9 cutting a
completely different gene thats very important for your body to function.
Second, a high amount or concentration of Cas9 can contribute to off-target activity. In
CRISPR gene therapy, genes that code for Cas9 are introduced into a cell instead of the genes
that would be directly introduced. Certain modes/methods of delivery can influence the amount
of time that these genes produce Cas9, which can affect the amount of the nuclease that is
present. For example, viruses are sometimes used to deliver genes to cells. Some viruses, such as
adenoviruses, causes the genes to be replicated many times by the DNA. This ultimately leads to
a longer expression of Cas9, resulting in a higher rate of off-target activity.

Making the Cut

All of these factors and issues may sound scary to some extent, but there is much hope
for the future. Currently, researchers are working tirelessly to increase the specificity, or
accuracy, or Cas9. Here are two discoveries that were made recently.
Ex-vivo editing is a mode of delivery where cells are edited outside of the patient, as
opposed to in-vivo editing, where cells are directly edited while they are inside the patient. How
exactly can this help us? According to Cox et. al, ex-vivo editing allows whoever is editing the
cells to control the dosage. This will help control the concentration of Cas9, which, as mentioned
earlier, affects off-target activity. This mode of delivery also helps researchers oversee the editing
process easier so that they can see what goes right and what goes wrong and how much Cas9 is
needed in order to avoid off-target edits.
Another way that off-target activity can be reduced is by using a variant of Cas9 called
SpCas9. This mutant is found in Streptococcus pyogenes bacteria and has been shown to have a
much higher specificity than your regular Cas9. The crazy thing is, scientists have created
several variations of SpCas9 that are even more specific than the wild type! For example, a
mutant called SpCas9-HF1 can completely or nearly completely reduce off-target mutations that
occur across a range of different frequencies with wild-type SpCas9 to levels generally
undetectable by GUIDE-seq and targeted deep sequencing, according to Kleinstever et. al. This
means that this mutant has a lower chance of making any off-target cuts, as the experiment
described above has found that off-target activity is nearly undetectable by a program that detects
off-target cuts (GUIDEseq). Another mutant called SpCas9-HF4 increases on-target activity.
Kleinstever et. al has shown that these variations are pretty consistent and do not create any new
off-target sites.
So how do these variations work? There are two grooves of a CRISPR-Cas9 system
called the HNH and RuvC grooves, which are needed for stable strand separation.The charges of
these grooves are altered in Cas9 variations, allowing it to bind to a non-target strand of DNA
and keep it from interacting with the gRNA and the target strand. This is just part of the
biochemistry that makes this system work like it does.
The Future of CRISPR

CRISPR carries much potential for being a miracle for many who are in need of a cure.
But unfortunately, this technology is far from perfect. However, with all of the research and
innovations being done today, we may have a CRISPR-Cas9 system ready for humans within the
next several years.

CRISPR has the potential to edit out diseases from the human genome and create superhumans. (The
Economist)