That's the only page that I hadn't already torn out of the workbook. For a while I had a lot of my homework around; I don't know where it is now, somewhere in the boxes that I haven't bothered to unpack since moving to the apartment I have had for almost a year.
________________________________
Name: Lena
Kochman
Article Assignment 3 (60pts). Cite
sources INLINE. Wikipedia is NOT a source.
Figure 2.
Panel A. (4pts) The authors perform two
controls: 1. Targeting other areas in the EMX1 gene and 2. Targeting PAVLB and
Th genes. What was the purpose of these controls in this experiment?
One of the things that these researchers
were trying to do was to determine the number of applications of RNA-guided
genome editing. They increased the
number of areas that they were targeting to help determine that number and they
also contrasted the efficacy of the CRISPR technology that they had already
used with a chimeric, engineered RNA complex.
(p. 2, Cong)
Panel B.
a.
(2pts) What does chimeric RNA mean?
From more than one
species.
b.
(1pts) What do the authors combine to make their
chimeric RNA?
Human cells and
mouse cells.
c.
(1pt) What do they call their new chimeric RNA?
chiRNA.
Panel C.
a.
(2pts) How many locations on the EMX1 gene did they
test?
5.
b.
(2pts) Did the chimeric RNA work better than crRNA in
all situations? Explain:
The chimeric RNA did
not work as well as the crRNA for 4 of the 5 situations. Either the bands that were produced in the
crRNA tests were not as pronounced as the bands produced in the chiRNA tests,
or they were absent in the chiRNA tests.
The only exception was for the 5th protospacer, at which the
bands were comparable.
c.
(2pts) What is the control in this experiment?
SURVEYOR Nuclease.
d.
(2pts) Based on this experiment, which combination of
RNA and target position work the best?
crRNA and the first
target.
Figure 3. In this figure, the authors compare their method
(CRISPR/Cas9) with TALENS. Answer the following questions about Figure 3:
Panel A:
a.
(1pt) What is a point mutation?
“Point mutation, change within a gene in which one base pair in the DNA
sequence is altered.”
https://www.britannica.com/science/point-mutation
b.
(1pt) In M2, where is the point mutation located?
Where the red U is:
(p. 3, Cong)
Panel B:
a.
(2pts) Did any of the mismatch guides result in indels?
If so, which ones?
M17, M15, M13.
Panel C:
a.
(4pts) What is TALENS and how does it work?
“Both TALEN, which is comprised of a
pair of DNA binding proteins fused to the FokI nuclease, and CRISPR, which is a
complex between the Cas9 nuclease and a target-specific single guide RNA
(sgRNA), can edit DNA through either HR or NHEJ.”
http://www.genecopoeia.com/resource/genome-editing-talen-or-crispr/
Panel D:
a.
(2pts) Did the TALENS result in indels? If so, what was
the indel frequency?
TALENS did result in
indels, at percentages of 3.6, 3.8 and 4.5.
b.
(4pts) Compared to chimeric RNA and crRNA, did the
TALENS work better or worse? Which method of the three worked best? How do you
know?
The chimeric RNA
worked better than the TALENS, and the crRNA worked better than the chimeric
RNA. The respective indel percentages
for the chimeric RNA were 11, 7.4 and 8.6, contrasted to the TALENS percentages
of 3.6, 3.8 and 4.5. The indel
percentages for the crRNA were 22, 29 and 25.
c.
(4pts) In your own words, explain why it is important
to perform the experiment comparing CRISPR and TALENS. (Why is it an important
control?)
You want to know
which method works the best. The
Genecopoeia website says that “off-target activity appears to be less of an
issue for TALEN” than for CRISPRs. It
could be that TALENS are less adept at nicking DNA and creating double stranded
breaks in several locations at once, but this also could mean that they are
less likely to create unintended mutations than CRISPRs at this time, before
CRISPRs are developed to have better specificity.
http://www.genecopoeia.com/resource/genome-editing-talen-or-crispr/
Figure 4.
Panel A:
a.
(4pts) In your own words, compare and contrast NHEJ
(non-homologous end joining) with HDR (Homology Directed Repair) Note: HDR can
also be called HR (homologous recombination)
NHEJ doesn’t need a
gene template to repair broken strands of DNA, and HR does.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758668/
For (b.) and (c.), fill in the
blanks about SpCas9 and SpCas9n activity:
b.
(3pts) SpCas9 is a DNA
nucle-ase, which cuts 2 strand(s) of DNA and results in _____NHEJ or HDR repair.
c.
(3pts) SpCas9n is a DNA nick-ase, which cuts 1 strand(s)
of DNA and results in HDR or sometimes
NHEJ repair.
This is the text
from the article that seems relevant to this question:
The
wild-type SpCas9 is able to mediate site-specific DSBs, which can be repaired
through either NHEJ or homology-directed repair (HDR). We engineered an
aspartate-toalanine substitution (D10A) in the RuvC I domain of SpCas9 to
convert the nuclease into a DNA nickase (SpCas9n, Fig. 4A) (12, 13, 20),
because nicked genomic DNA is typically repaired either seamlessly or through
high-fidelity HDR. SURVEYOR (Fig. 4B) and sequencing of 327 amplicons did not
detect any indels induced by SpCas9n. However, nicked DNA can in rare cases be
processed via a DSB intermediate and result in a NHEJ event (27). We then
tested Cas9mediated HDR at the same EMX1 locus with a homology repair template
to introduce a pair of restriction sites near the protospacer (Fig. 4C). SpCas9
and SpCas9n catalyzed integration of the repair template into EMX1 locus at
similar levels (Fig. 4D), which we further verified via Sanger sequencing (Fig.
4E). These results demonstrate the utility of CRISPR for facilitating targeted
genomic insertions. Giventhe 14-bp (12 bp fromthe seed sequence and 2 bp from
PAM) target specificity (Fig. 3B) of the wild-type SpCas9, the use of a nickase
may reduce off-target mutations.
(p.
4, Cong)
Panel B:
a.
(2pts) In this experiment, is the chimeric RNA a
constant or a variable? Explain how you know this.
The chimeric RNA is
a constant because it’s in every well.
b.
(2pts) Did SpCas9 or SpCas9n work better in this
experiment? Explain how you know this.
The SpCas9 worked
better. It resulted in indel percentages
of 5.3, 4.6 and 3.5 for the first three wells, respectively. No other indel percentages are listed, so it
seems as if no other well had them. The
first three wells all had SpCas9 and did not have SpCas9n.
c.
(6pts) In your own words: Explain why, for the CRISPR
system to work, the cell needs to rely on NHEJ repair.
The double-stranded
breaks need to be repaired after they are effected by the CRISPR.
This is a quote from
an article called “DNA double strand break repair via non-homologous
end-joining”:
”
CRISPR is ultimately
going to be used as part of gene therapy, which can’t be dependent on being
administered at such precise times as are required by the cell’s manufacture of
nucleotide templates.
Panel F:
a.
(2pts) How many genes is this new construct targeting?
2.
b.
(1pt) What are the names of these genes?
EMX1, PVaLB.
c.
(3pts) Starting from Left to Right, number the lanes 1-8
in this figure. Which lanes are the controls and which lanes are the
experimental condition?
7 and 8 are the
controls; the rest are experimental.
________________________________________________
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