Inquiry Question: How has CRISPR-Cas9 treatment opened new avenues in both the progress and complications of editing

the
mutation causing Leber's congenital amaurosis 10?
Scientists have been able to use genetic technologies to 'cut and paste' DNA for decades. However, the newest technology,
CRISPR Cas9 aims to revolutionise treatments of cancers and rare genetic diseases such as Leber's Congenital Amaurosis 10
(LCA10.This powerful new tool; CRISPR Cas9 has opened new avenues in genomic editing as it can delete the mutated CEP290
gene, causing the vision loss that characterises LCA10, and add the corrected version with more precision than ever before.
Although its only use in clinical trials for LCA10 suggests the uncertainties around CRISPR remain high. Hence, this article seeks
to evaluate the progress and complications CRISPR Cas9 has in the treatment of LCA10.

Disease Profile: Leber congenital amaurosis (LCA10) is a genetic eye disease that primarily affects the retina. People with this
disorder typically have severe visual impairment beginning in infancy as their eyes can no longer perceive light. The biological
cause of this disease is the nonsense mutation of the CEP290 gene which acts as a stop signal, halting the production of the
protein necessary for the light-sensing cells photoreceptor cells in the retina, hence causing vision loss. Additionally, LCA10 has
autosomal recessive inheritance, hence, if both parents are carriers or possess the disease, it is possible for their offspring to
have LCA10. This allows, the disease to be prevented through genetic testing before pregnancy which identifies whether the
embryo acquired the mutated gene. Management protocol for affected individuals include procedure to correct refractive error or
use of vision aids when possible. However, LCA10 is currently treated using gene therapy but the most recent technology,
CRISPR Cas9 is a treatment involving the cutting and replacing of the mutated gene with a functional gene, allowing the
production of the protein crucial to the photoreceptor.
Referring the graph 1.1:
 Trend 1: As the number of days since (data is collected) increases from day 0 to day 6, the expression of wild type (WT)
mRNA increases dramatically (greatly) from 107.5μg to 109μg. Similarly, in the same increase of days, the expression of gRNA from
the edited CEP290 increases dramatically at 10 6.5μg to 108μg, a slightly faster rate than the WT. This is because the editing by
CRISPR Cas9 to CEP290 causes a sudden significant increase in the expression of gRNA which was initially relatively little. In
comparison to the wild type who continually expressed mRNA for this gene.
Trend 2: The expression of mRNA remains consistently higher, plateauing between 10 8.5μg and 109μg, than the expression of
gRNA which plateaus at approximately 10 8.1μg, as weeks from post editing increase. This is because wild type patients don't
possess the disease and thus have a pre-existing higher expression of corrected CEP290 as RNA. The similar maximums
between gRNA and mRNA suggests the successfulness of CRISPR Cas9 in achieving expression levels similar to the those
without the disease; the WT.
MAKE MORE RELEVENT TO PROGRESS/COMPLICATION = INQUIRY QUESTION LINK  COULD ADD OLD GRAPH WITH
THE “3RD” TREND
Use of Technology:
The CRISPR Cas9 technology effectively treats the LCA10 disease as the repaired corrected CEP290 gene can now be
transcribed into the normal mRNA and thus is further translated into the fully functional CEP290 protein required in the structure of
photoreceptor cells. Diagram 1.2 shows how the protein formed from the expression of the normal CEP290 forms the connecting
cilium in the photoreceptor cell. Whereas, the previously mutant CEP290's stop signal didn't allow a full connecting cilium protein,
and hence, a dysfunctional photoreceptor.

Discussion of Technology:
Benefits Limitations

Conclusion: Hence, this inquiry observed the ability of CRISPR Cas9 in treating Leber's Congenital Amaurosis through its ability to
accurately cut and replace the mutated CEP290 gene to produce the structural protein required to restore vision. Through
evaluation of CRISPR Cas9, it was found to be beneficial through its ability to improve the ease and efficiency of treatment in
comparison to pre-existing treatments, and its potential use in preventinLCA10 in children. Opposingly, it has raised complications
of social fears of off target affects in such a new experimental technology and its potential to foster economic inequality due to the
expensive nature of the treatment. Regardless, CRISPR Cas9 has proved to be a powerful tool that leads to future avenues of
research for greater precision and efficiency; where researchers aim for simultaneous editing, where a Cas12a is created which
will enable CRISPR to edit many genes in a short space of time and thus improve the gap between the expression of the
corrected CEP290 gene in comparison to non-affected individuals' (wild type) expression of this gene. This can be further
improved in the future through research of CRISPR Cas9 modifying gene expression, where this can be increased to express
more CEP290 in LCA10 patients, and thus improve their rate of expression of the corrected gene, hence treating the disease
efficiently.