Cut-It-Out!

CRISPR case study

Part I. TED talk (David, grandfather, retired civil engineer)

David paused as he walked towards his favorite reading chair, absorbing the early morning
sunlight peaking through the window. Even though he was retired now, he still woke early and
liked to get started on his day. Rather than sitting in an office chair working on draft plans for a
new community center, he now began his day here in his living room with his iPad. He had
adjusted to this change in accessing information and appreciated that one didn’t have to wait
for the daily paper to see what was going on in the world. After a quick scan of the headlines -
which revealed nothing too interesting - David navigated to the TED website. He had recently
become hooked on TED Talks, and relied on these much more than the Discovery or History
Channel to help him keep learning. In another life, he would have been a college professor…

He had just finished watching the 2015 best science talks playlist yesterday (a 16 year old
developing a test for pancreatic cancer? wow!), so what next? What about genetics? What
was new with all that human genome research? And wasn’t his granddaughter Nadia taking a
genetics class this term at UCLA? She’d be home for the holidays soon...maybe he could
impress her with his up-to-date knowledge.

Scrolling through his search results, a name caught his eye: Jennifer, his late wife’s name. What
was this about - editing our DNA? Well, it must be fate - Jenny was a writer and editor, so this
should be a good place to start his morning.

Fifteen minutes later, as the closing applause signified the end of the video, David shook his
head. Bacteria’s defense against viruses to curing human disease? This is why he had been a
civil engineer. Living things were so much more complicated....

Questions: Worth 8 points total

1. Where did the CRISPR system first originate in nature and what is its purpose in that context?
Worth 2 points

The CRISPR system first originated in nature when a virus infected a bacteria cell with its own
DNA and the CRISPR system records the sequence of DNA from the virus before destroying it. In
this context CRISPR essentially acts like an immune system by recording viral DNA sequences
and saving them for future attack. The CRISPR system also passes down those sequences to
future generations to protect other cells. The cell then creates 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? Worth 2 points

Cas9 then 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. Basically, the CRISPR system first takes the DNA and
makes an RNA copy of it, then Cas9 bins to that RNA sequence and uses that same sequence to
find the site that matches it in the DNA, and finally destroys that particular DNA sequence

3. What advantage does the CRISPR/Cas9 system offer compared to previous genome editing
technologies? Worth 2 points

CRISPR/Cas9 system 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.
Therefore, 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 a
repair in DNA.

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
models systems an important part of research? What might David tell his friend about basic
research, now that he’s seen this TED video? Worth 2 points

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. Also, it is 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 CRISPR/Cas9 system is usually used in bacteria this technology has to be
adapted and adjusted to work in human DNA. David could talk about how since scientists has to
find the best way to transfer CRISPR/Cas9 system into the cell, how to prevent CRISPR/Cas9
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 most likely test different variables to make sure the
technology is safe as it possibly can be before allowing clinical research to take place.

Part II. Clinic (Audrey, Nadia’s aunt, a carrier for Duchenne Muscular Dystrophy, DMD)

Audrey and her partner Andrew sat in the corner of the diner, trying to process the information
they had received during their meeting with the team from the research hospital. Many
couples planning for a pregnancy have anxieties and questions, but this pair felt they had more
than most. During her teenage years, shortness of breath had led to genetic tests that revealed
Audrey was heterozygous for a mutation in the dystrophin gene. The dystrophin gene is X-
linked; since Audrey has a functional allele on her other X chromosome, the impacts of her
health have been minimal. But now she and Andrew would like to have children and they are
concerned about the possibility of having a son with Duchenne muscular dystrophy (DMD), a
disorder that results when no functional dystrophin is made.

At their first meeting with a genetic counselor, they had talked about in vitro fertilization (IVF)
and pre-implantation diagnosis (PGD), which would allow them to select only female embryos
or embryos that did not carry the altered dystrophin allele. They understood that this would
allow for genetic screening of embryos and then a decision would have to be made about which
embryo(s) to transfer for Audrey’s uterus hopefully for successful implantation. But then one
of Audrey’s friends on Facebook highlighted a report about a new technology that helped mice
with muscular dystrophy and the couple realized they had more questions about their possible
options. Audrey just wishes she had been able to schedule this meeting when her niece Nadia
was visiting for Thanksgiving. Nadia was studying biology in college and had discussed the
genetics underlying this situation with her aunt before. The medical team had shared with the
couple the possibility of using new genome editing technology to modify the dystrophin gene in
fertilized eggs or even to treat an individual diagnosed with DMD after birth. What was the
name of this method? Andrew dug through his backpack and pulled out his notes - CRISPR/Cas,
it is called - and Audrey texted Nadia to ask about it.

Questions: Worth 8 points total

1. Why is the couple concerned about having a son with DMD - are their daughters not at risk?
Worth 2 points

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

individual that lacks this allele, has been explored as a clinical approach for decades. Why is
the CRISPR/Cas system more favorable compared to this conventional type of therapy?
Worth 2 points

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. However, 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.
3. What challenges might there be in using genome editing to treat an individual diagnosed
with DMD after birth? Worth 2 points

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.

4. 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? Worth 2 points

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. Undergraduate science class (Nadia, granddaughter, an undergraduate student)

Nadia sat in her biology class with a box of tissues in front of her. She felt miserable and her
doctor told her that it was a viral infection. Since antibiotics wouldn’t affect a viral infection,
she was just going to have to let her immune system fight it off! She reached for another
tissue when her instructor began talking about prokaryotes and how they have a simple
adaptive immune system. Nadia perked up and wondered, “How could a bacteria or archaea
have an immune system...immune systems are really complex!” She thought about her own
illness...she figured her body was recognizing the foreign virus and launching immune cells to
target and destroy it. She always imagined an immune attack like a Star Wars space battle.

Since prokaryotes are single cells, Nadia began to listen to her professor explain how a single
cell can build up defenses against a viral attack. She copied down the reference her professor
listed on the slide [Sampson, T.R. and Weiss, D.S. Exploiting CRISPR/Cas systems for
biotechnology. Bioessays 36 (2014): 34-38] and planned to take a look at the first three sections
that her professor had highlighted earlier in class: the abstract, introduction, and the section
titled, “Cas9 as a programmable DNA targeting platform”.

When Nadia got out of class, she took a quick glance at her phone as she was headed to
lunch. A text from her aunt Audrey read: “Do you know anything about CRISPR/Cas
technology? We meet with the medical team today and they said it was a possible treatment
option for us.” “Whoa!” Nadia thought, that was the method that had been brought up in class
today! She settled into a corner table in the cafeteria and flipped open her laptop to look up
the reference.

Questions: Worth 7 points

The basics
1. Based on your understanding of nucleic acids, what type of bonds would form between the
CRISPR molecule and the target DNA? What type of bonds would an enzyme such as Cas
affect? Worth 2 points

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.

2. What is meant by “off-target effects”? Why do these occur? Worth 2 points

Off target effects refers to side effects or unintended effects. These effects are not intended
for when using the medicine and the patient must be aware of these effects so they know if
a drug is severely, negatively affecting their body. These occur in many different drugs
because drugs can sometimes change the shape of protein or a substrate which effects the
functions in the body.

DNA repair: Figure 1 shows two types of DNA repair, non-homologous end joining (NHEJ) and
the other is 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? Worth 1 pt

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? Worth 2 points

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. Research

Nadia found herself so intrigued by this technology - and all its possible applications - that she
almost missed her afternoon class! After a nap and dinner, she was feeling slightly better and
decided to look for more information specifically about CRISPR/Cas and DMD, so she could be
helpful to her aunt. Her aunt had shared with her the news article about how the CRISPR/Cas
technology had helped mice with muscular dystrophy so Nadia knew that looking at the
primary literature the news article was based on would help her understand the
research. Nadia read in the news article that the information was based on three different
primary papers published back-to-back in the 22 January 2016 issue of the journal Science. She
pulled up the three articles and began examining the report by Nelson et al., reading through
the abstract, introduction and the results depicted in Fig. 1.

Questions: Worth 19 points

1. What is the name of the primary article that demonstrates the work of the Gersbach lab?
Worth 1 point

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular
dystrophy

2. The link Nadia got from her aunt is a secondary article. The three groups whose research is
discussed in the secondary article published their work directly in primary literature
articles. Access each of the three full articles and compare their general contents to each other
and the secondary news article. What are some components you consistently see in scientific
primary literature papers that distinguish them from secondary articles? Worth 2 points

The primary literature papers are different from the secondary papers all have a reference
section and notes section. This section of the paper was most likely a key component of the
paper because it adds credibility to the paper since the target audience is most likely an
academic audience. Adding the reference section also allows others reading the paper to look
more into the subject by citing the sources in the body. The literature papers also thoroughly
explain the procedure of their experiments. This was added so others reading the paper would
be able to repeat the experiment to see if they have the same results as the papers. Being able
to repeat the experiment and getting the same results adds more credibility to the hypothesis.
Hypotheses was another key component the primary literature papers had which basically told
the reader what the scientists wanted to support or refute based on their experimental results.

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? Worth 2 points

The mdx mouse model from the article has a nonsense mutation in exon 23. A nonsense
mutation codes for a stop codon before a protein is completely formed and this will change the
size of the protein because less amino acids will be used to form a protein; therefore the
protein will not function correctly. Since removing exon 23 will remove the mutation from the
cell, the article states that this will help improve the contractility of muscles. Thinking about this
biologically, this shows that actin filaments most likely are improving around muscle cells to
improve contraction in muscles.

4. In your own words, use Figure 1 A to describe how this group used the CRISPR/Cas system to
“correct” this mutation? Worth 3 points

Cas9 nuclease is an enzyme that cuts up DNA sequences by using certain RNA sequences to
locate the correct DNA sequence to cut. In Figure 1A the Cas9 nuclease uses two single guide
RNA that each have the sequence for intron 22 and 23. The Cas9 nuclease looks for this
sequence in the DNA and then breaks the hydrogen bonds between the double stranded DNA
(like a pair of scissors) at this region which includes the mutated exon 23. Then the DNA is
repaired by NHEJ so protein production can continue without the mutation.

5. Nadia thought back to the article she read for class and thought, “They could have done that
a different way though!” Still using the CRISPR-Cas system, what could they have done differently
in the mdx mouse to create a functional dystrophin protein product? Worth 2 points

Instead of repairing the protein by NHEJ which can cause frameshift mutation a donor construct
could have been used to repair the DNA sequence by homology directed repair. However,
according to the scientific report HDR is not as frequent in cells that are not replicating so this
would more useful for cells that can still replicate.

6. In Fig 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.
Worth 3 points.

RT-PCR is used to measure mRNA by amplifying it after converting it to

complementary DNA. This can be used to check if the genome editing worked by
amplifying exon 23 sequence and seeing if the gene is expressed afterwards. If the
cells expressed show defected protein then that means the exon 23 sequence was
not removed but if it is not expressed then it was removed from the sequence.

b. Explain why there are multiple bands in the treated lanes and how that links to Fig
1E? Worth 2 points

There are multiple bands in the treated lanes because the multiple bands towards
the bottom of the picture represent DNA that was cut and was able to migrate
future away from the wells from the electrophoresis gel. The migrated bands
represent the exon 23 DNA sequence. Figure 1E shows that 59% of the products
after treating with CRISPR/Cas9 system do not have exon 23. This coincides with the
multiple bands in the treated lanes because it shows that the DNA was cut at a
 certain point therefore not part of the DNA anymore.

7. Given the results of this study, as stated in the abstract, 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/Cas approach to treat DMD post-natally? Worth 4 points

For the strengths Nadia can tell her aunt that CRISPR/CAS approach works on 83% of patients
with DMD. This technique is also more efficient than trying to replace the entire defective DMD
gene. This technique was also shown to have little to no evidence of off target effects when
tested in three different research groups. One limitation was that the technique described in
Gersbach’s lab had to use NJEH which can cause frame shift mutations because certain cells
that are stuck in the G0 phase can no longer divide. Therefore, while the three research groups
did not find a lot of evidence of off target effects it does not mean they do not exist. Another
limitation is that the mice with DMD did not function as well as mice without DMD so this
genome editing technique is not a cure to DMD. Since the effects of CRISPR could fade over
time then the treatment might just delay DMD from progressing quickly but it would still
continue to degrade muscle cells over time.