Ellie Seamons

BIOL 1210 001

Part I
1. Where did the CRISPR system first originate in nature and what is its purpose in that
context?
When a virus infected a bacteria cell with its own DNA. The CRISPR system
recorded the sequence of DNA from the virus before destroying it. CRISPR acted like an
immune system by recording viral DNA sequences and saving them for future attack.
CRISPR also passed down those sequences to future generations to protect other cells.
The cell would then continue to create an RNA copy of the DNA

2. How is Cas9 able to bind to specific sequences of DNA? What does Cas9 do to the
sequences it interacts with?
Cas9 uses the pieces of RNA to bind to, and then tries to find that same sequence
in the DNA to cut up the piece of viral DNA. The CRISPR system first takes the DNA
and makes an RNA copy of it, then Cas9 binds to that RNA sequence and uses that same
sequence to find the site that matches it in the DNA, and then destroys that particular
DNA sequence.

3. What advantage does the CRISPR-Cas9 system offer compared to previous genome
editing technologies?
It is simpler compared to older genome editing technology because it is
programmable so, if there is a piece of DNA that is mutated, a scientist can program the
CRISPR/Cas9 system to target that specific DNA sequence using a small piece of RNA.
That piece of DNA sequence will be cut and should trigger the cells natural ability to
repair that mutation. So, scientists use little manipulation to help repair mutated DNA
because they are just using the CRISPR/Cas9 system to create a double stranded break in
the DNA that will trigger the DNA to repair itself.

4. David remembers one of his friends discussing how the government should focus
funding more on clinical research rather than basic research. His friend felt that basic
research in model organisms was not very important anymore since we know so much
about genetics. Why are model systems an important part of research? What might David
tell his friend about basic research, now that he’s seen this TED video?
Please refer to the TED video to answer this question:
(<http://www.ted.com/talks/jennifer_doudna_we_can_now_edit_our_dna_but_let
_s_do_it_wisely>)
David might tell his friend that new technology should be tested on model
organisms first because they are easier to reproduce and grow in the lab. Using model
organisms will allow the scientists to create various trials and experiments to study the
effect of various variables on the results. It is also much easier to control different
variables such as diet and amount of sleep in a model organism because they are grown
and taken care of in the lab so scientists control many more factors than they would be
able to when using humans to test out new technologies. Since this system is usually used
in bacteria, this technology needs to be adapted and adjusted to work in human DNA.
David should talk about how since scientists has to find the best way to transfer
CRISPR/Cas9 system into the cell, to prevent the system from cutting untargeted DNA
sequences, how to control the way the DNA is repaired, and what side effects can arise
from using CRISPR/Cas9 and how to prevent them. Since there are so many questions to
answer, scientists will probably test different variables to make sure the technology is as
safe as it possibly can be before allowing the clinical research to take place

Part II
1. Why is the couple concerned about having a son with DMD? Are their daughters not at
risk?
The daughter will only be a carrier if Andrew does not carry the gene for DMD
and according to the story it appears only Audrey carries the gene for DMD. Since the
daughter must inherit the dominant X chromosome from her father she will be unaffected
by DMD. However, if Audrey has a son, there is a 50% chance her son could have DMD
since he would receive a Y chromosome from Andrew and one of the X chromosomes
from Audrey. Since one of Audrey’s X chromosomes has the recessive allele for DMD,
her son will have DMD if he receives that allele.

2. Nadia’s mother Rachel is Audrey’s biological sister. Should Nadia herself be

concerned about being a carrier of the DMD allele or having the disorder?
Since Audrey is a carrier, there is a possibility that Rachel is too. Nadia should
only be worried if she has DMD if her father was also a carrier. If her father was not a
carrier, then there is a 75% possibility that she isn’t either. Because she inherited the X
chromosome from her father, however, and she hasn’t shown symptoms of the disorder,
there is only a 25% chance she will be a carrier. She could be safe and get genetically
tested, but if she marries someone who isn’t a carrier, her child will have even less of a
chance.

3. Conventional gene therapy, described as introducing a functional copy of an allele into

an individual that lacks this allele, has been explored as a clinical approach for decades.
Why is the CRISPR-Cas9 system more favorable compared to this conventional type of
therapy?
Introducing a functional copy of a gene is not as effective as CRISPR/Cas9
system because the entire gene is too large to be replaced despite the fact that scientists
wanted to use the CRISPR/Cas9 system to cut out an exon to shorten the gene for DMD.
By shortening the gene scientists found mice with DMD had gain more muscle strength
and the gene was repaired in muscle stem cells. The scientists also found that the
CRISPR/Cas9 system did not cut off-target sequences in the DNA. While this is only the
beginning of treatment, scientists believe that 80% of DMD patients can benefit from
removing an exon and this treatment has had positive effects in mice

4. what challenges might there be in using genome editing to treat an individual

diagnosed with DMD after birth?
 It would be challenging because after birth the cells have begun to differentiate
and many of them most likely have turned off certain genes so it would be difficult to
signal repair in a certain part of the body to regenerate new cells. As described in the
article, muscle cells in adults do not divide and lack the ability to repair DNA so if an
adult were seeking treatment, it would not help to edit the genome since many of the cells
are stuck in the G
0 phase. Another challenge would be immediately after birth, many of the baby’s cells
have already formed so results would not be immediate. While many of the cells are still
growing, they won’t all immediately replace the defective cells. It would take time but
there would be a possibility of repairing some of the defective tissue.

5. How might the way we think about using these gene editing technologies change if we
imagine them being employed for purposes other than clinical disease
prevention/treatment?
If someone were to use gene editing technologies to change their hair, skin, or eye
color, to have an advantage in athletics, or for other appearance changes this technology
might lose respect. For example, plastic surgery has negative connotations since many
people use plastic surgery to change their appearance despite the fact that plastic surgery
has also been used to help burn victims heal their scars. Therefore, gene editing
technology might receive the same negative connotation even if it is used for positive
effects.

Part III
The Basics
1. What does CRISPR stand for?
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat.

2. Based on your understanding of nucleic acids, what type of bonds form between the
CRISPR/guide RNA molecule and the target DNA? What type of bonds would an
enzyme such as Cas9 affect?
The CRISPR molecule would form hydrogen bonds with the target DNA because
DNA has a partial negative charge which allows attraction to a partial positive charge.
The enzyme Cas9 would affect the hydrogen bonds formed between the two strands by
breaking them apart.

DNA Repair
For this subsection, please refer to Figure 2 in the Zhang article, which shows two
types of DNA repair, non-homologous end joining (NHEJ) and homology directed repair
(HDR).

3. When Cas9 induces cleavage resulting in NHEJ, it causes mutations in the cell. One
common effect is a frameshift mutation: how does this affect gene expression?
A frame shift mutation affects gene expressions because it shifts the entire frame
of coding either up or down. When an amino acid is inserted the sequence shifts up by
one base pair and the codons will code for different amino acids or a deleted amino acid,
such as in this case, will shift the entire sequence down by one base pair and the codons
will code for different amino acids as well. Coding for different amino acids will cause
different proteins to be made since amino acids are the building blocks of proteins.

4. How is HDR different and why would this be desirable?

HDR does not remove sequences from both of the DNA strand but only removes
the sequence from one strand to nick the DNA. When a donor construct is added the
DNA becomes repaired which reduces the risk of a frameshift mutation because only one
strand of DNA sequence is cleaved rather than both. If both strands were cleaved then
DNA repair must occur and this opens the DNA to deletion or insertion of nucleotides.
However, with HDR a donor construct already exists and is just added to the nick to
repair the DNA.

Part IV
For this section, please refer to the Nelson et al. article and read through the
Introduction and the results depicted in Figure 1.

2. We will focus here on the study published by Nelson et al. What is the name of that
primary article? Why do you think these three articles were published “back-to-back”?
The primary article is called “In vivo genome editing improves muscle function in
a mouse model of Duchenne muscular dystrophy”. I think they were published back-to-
back because they build upon each other and provide more information to the previous
article, they go a little more in-depth than the primary article.

3. From the Nelson et al. article, state what type of mutation the mdx mouse carries and
describe the result of such a mutation in terms of the final protein product.
The MDX mouse carries the AAD-Dmd strand and the CRISPR treatment
resulted in partially recovered muscle functional deficiencies and generated a pool of
endogenously corrected myogenic precursors.

4. In your own words, use Figure 1A to describe how this group used the CRISPR-Cas9
system to “correct” this mutation.
This group used the CRISPR-Cas9 system to correct this mutation by mediating
the genomic and transcript deletion process of the exon 23 through the process of
intramuscular AAC-CRISPR administration.

*5. Nadia thought back to the article she read for class: They could have done that a
different way though! What could the researchers have done instead to create a functional
dystrophin protein product?
They could have used HDR so that the DNA would have a reduced chance of
replicating the mutation.
*6. In Figure 1D, they do an assay called RT-PCR. Look up this method and then in your
own words: a. Describe how this assay can be used to determine if their genome editing
worked. b. Explain why there are multiple bands in the treated lanes and how that links to
Figure 1E.
a. RT-PCR makes complementary DNA fragments from RNA, effectively reverse
transcribing specific genes that are expressed. If the edited genes are functional, then the
RR-PCR will make it possible to quantify them, as those genes whicha re functional will
have fragmented clones.
b. The treated lanes have multiple bands, because they have had fragments cut
from the rest of the DNA, resulting in more differentiated lengths of fragments which
will get separated during the process of gel electrophoresis. 1E reflects upon this,
showing that about 58 percent of the DNA does not contain the exon 23, all due to
CRISPR/Cas9

7. What are the implications of this result for restoring muscle function?
The disorder is a whole lot less likely to appear due to CRISPR/Cas9. The
treatment would be beneficial to try, but it comes with its own set of risks that Audrey
and her husband would need to discuss to see if the benefits outweighed the risks.

8. Given this data and other considerations raised in this case study, what might Nadia
share with her aunt about the current strengths and/or limitations of using a CRISPR-
Cas9 approach to treat DMD postnatally?
Nadia should tell Audrey that CRISPR/Cas9 approach is one of the most precise
in treating DMD, allowing for complete removal of mutated genes, but it can have
potentially dangerous effects in the event of a mistake. While this method has the
potential to alleviate the reduced production of dystrophin, it may also make cuts that are
off target. While this is rare, it could have harmful effects on the baby it’s inserted into.