BCH5413 TEST 2 ACTUAL EXAM SCRIPT
2026 FULL QUESTIONS AND CORRECT
ANSWERS REVISED EDITION
⩥ When creating a knockout animal, why do you need to engineer ES
cells? Answer: **Able to perform manipulation of the genome but the
cells still grow into a whole organism. ES cells give rise to all body
tissue types. iPS cells: take cells that are differentiated and cause them
too undifferentiated.
**ES cells = pluripotent, used to knock out genes of interest.
⩥ When creating knockout ES cells, why do you need to isolate cells
that are sensitive to G418 AND ganciclovir? Answer: To tell you where
your transgene is being expressed!
G418 neomycin analog kills cell without neomycin-resistance gene
(cells with no integration)
Gangcyclovir kills cells containing thymidine kinase gene (cells with
non-specific recombination)
Cells that grow on medium will have homologous recombination
between the plasmid and WT gene
,⩥ When creating a knockout mouse, the chimeric animal is mated with
one that is black and normal for the gene of interest. From this cross,
three possible offspring can be produced. Explain the genetics of each of
the three and what you would do next with the desired offspring.
Answer: possible progeny genomes include: A/X+ (brown, no gene of
interest), A/X- (brown, gene of interest), a/X+ (black, no gene of
interest) in which A/X- is the most desirable genome.
I would then mate two heterozygous brown mice (X+X-) containing the
gene of interest to produce a homozygous brown knockout mouse (X-X-
) and screen by southern blot.
⩥ Where in the genome are loxP sites located in a Cre/Lox model?
a. Adjacent to the Cre locus expressed in a desired cell type
b. On either side of the gene that is to be knocked out
c. Random sites throughout the genome Answer: b. On either side of the
gene that is to be knocked out
⩥ In which organism was the CRISPR system discovered? Answer:
System of bacterial natural immunity. From studying natural immunity
in bacteria.
⩥ In the immunization phase, what happens to the foreign viral DNA?
Answer: Phase 1(immunization phase): how the bacterial cell makes
itself immune
Phase 2 (immunity phase): how it uses that to protect itself from a
second infection.
,The foreing viral DNA is cleaved into smaller pieces called spacers. The
spacers are incorporated into the CRISPR locus; part of that region of
the chromosome has the spacers in it. The small pieces of viral DNA are
separated from each other by black boxes called repeats.
⩥ In the immunity phase, what RNAs are present in the Cas9 complex?
And what is their role? Answer: pre-CRISPR RNA-contains the spacers
and repeats
RNase III-cleave the RNase between the spacer and the spacer and
repeat, cleaves between these spacers and repeats from the rest of the
spacers and repeats
tracrRNA-tracer binds to the repeat, which makes a recognition site for
the RNase III
⩥ What happens in a new infection from a previously recognized viral
DNA? Answer: The second time the bacteria encounter the same viral
RNA, it will go into the cell and it will complementary base pair with
the spacer because it was made from the same viral DNA. The viral
DNA will then get cleaved by the CRISPRS
⩥ What is the importance of PAM sequence in CRISPR Cas9
technology? Answer: THE PAM sequence is present in the invading
viral DNA, and it is not present as part of the spacer. So the cell or the
cas9 complex recognizes that the viral DNA is non-self and it will get
cleaved.
, ⩥ What are the 2 possible repair mechanisms of a double strand break?
Which one of the methods would you prefer if you wish to make a three
nucleotide change in your target gene using CRISPR Cas9? Answer: The
first mechanism is to leave the double strand break alone and in some
number of cells you would get non-homologous end joining. During this
process you get insertions and deletions where the DNA was cleaved.
The gene is likely to become nonfunctional if non homologous end
joining occurs. Get a knockout of the gene of interest and allow non-
homologous end joining to occur.
The second mechanism is by using 2 target DNA, so 2 spacer regions
that are close by to each other so that the cas9 system will bind to both
regions. Allows for cleave on either side. Advantage: gives an offset
double stranded break instead of a straight double stranded break, this is
more efficient in homologous recombination.
⩥ In the Zinc Finger approach to gene editing, what would be the
equivalent of the guideRNA from CRISPR? Why? (explain the function
of each) Answer: Useful technology for gene editing, specifically for
HIV infection.
⩥ In the example given, what is the importance of the CCR5 gene?
Briefly describe how this technology is helpful in controlling the
replication of HIV-1. Answer: People who have a mutation of the CCR5
gene are resistant to HIV infection. The CCR5 creates a receptor on the
surface of the white blood cells and that's what HIV binds too. People
who have a natural mutation to this gene prevent HIV from binding,
mutation does not lead to any other symptoms. The zinc finger protein is
used to take an HIV infected person and cleave their DNA and allow it
2026 FULL QUESTIONS AND CORRECT
ANSWERS REVISED EDITION
⩥ When creating a knockout animal, why do you need to engineer ES
cells? Answer: **Able to perform manipulation of the genome but the
cells still grow into a whole organism. ES cells give rise to all body
tissue types. iPS cells: take cells that are differentiated and cause them
too undifferentiated.
**ES cells = pluripotent, used to knock out genes of interest.
⩥ When creating knockout ES cells, why do you need to isolate cells
that are sensitive to G418 AND ganciclovir? Answer: To tell you where
your transgene is being expressed!
G418 neomycin analog kills cell without neomycin-resistance gene
(cells with no integration)
Gangcyclovir kills cells containing thymidine kinase gene (cells with
non-specific recombination)
Cells that grow on medium will have homologous recombination
between the plasmid and WT gene
,⩥ When creating a knockout mouse, the chimeric animal is mated with
one that is black and normal for the gene of interest. From this cross,
three possible offspring can be produced. Explain the genetics of each of
the three and what you would do next with the desired offspring.
Answer: possible progeny genomes include: A/X+ (brown, no gene of
interest), A/X- (brown, gene of interest), a/X+ (black, no gene of
interest) in which A/X- is the most desirable genome.
I would then mate two heterozygous brown mice (X+X-) containing the
gene of interest to produce a homozygous brown knockout mouse (X-X-
) and screen by southern blot.
⩥ Where in the genome are loxP sites located in a Cre/Lox model?
a. Adjacent to the Cre locus expressed in a desired cell type
b. On either side of the gene that is to be knocked out
c. Random sites throughout the genome Answer: b. On either side of the
gene that is to be knocked out
⩥ In which organism was the CRISPR system discovered? Answer:
System of bacterial natural immunity. From studying natural immunity
in bacteria.
⩥ In the immunization phase, what happens to the foreign viral DNA?
Answer: Phase 1(immunization phase): how the bacterial cell makes
itself immune
Phase 2 (immunity phase): how it uses that to protect itself from a
second infection.
,The foreing viral DNA is cleaved into smaller pieces called spacers. The
spacers are incorporated into the CRISPR locus; part of that region of
the chromosome has the spacers in it. The small pieces of viral DNA are
separated from each other by black boxes called repeats.
⩥ In the immunity phase, what RNAs are present in the Cas9 complex?
And what is their role? Answer: pre-CRISPR RNA-contains the spacers
and repeats
RNase III-cleave the RNase between the spacer and the spacer and
repeat, cleaves between these spacers and repeats from the rest of the
spacers and repeats
tracrRNA-tracer binds to the repeat, which makes a recognition site for
the RNase III
⩥ What happens in a new infection from a previously recognized viral
DNA? Answer: The second time the bacteria encounter the same viral
RNA, it will go into the cell and it will complementary base pair with
the spacer because it was made from the same viral DNA. The viral
DNA will then get cleaved by the CRISPRS
⩥ What is the importance of PAM sequence in CRISPR Cas9
technology? Answer: THE PAM sequence is present in the invading
viral DNA, and it is not present as part of the spacer. So the cell or the
cas9 complex recognizes that the viral DNA is non-self and it will get
cleaved.
, ⩥ What are the 2 possible repair mechanisms of a double strand break?
Which one of the methods would you prefer if you wish to make a three
nucleotide change in your target gene using CRISPR Cas9? Answer: The
first mechanism is to leave the double strand break alone and in some
number of cells you would get non-homologous end joining. During this
process you get insertions and deletions where the DNA was cleaved.
The gene is likely to become nonfunctional if non homologous end
joining occurs. Get a knockout of the gene of interest and allow non-
homologous end joining to occur.
The second mechanism is by using 2 target DNA, so 2 spacer regions
that are close by to each other so that the cas9 system will bind to both
regions. Allows for cleave on either side. Advantage: gives an offset
double stranded break instead of a straight double stranded break, this is
more efficient in homologous recombination.
⩥ In the Zinc Finger approach to gene editing, what would be the
equivalent of the guideRNA from CRISPR? Why? (explain the function
of each) Answer: Useful technology for gene editing, specifically for
HIV infection.
⩥ In the example given, what is the importance of the CCR5 gene?
Briefly describe how this technology is helpful in controlling the
replication of HIV-1. Answer: People who have a mutation of the CCR5
gene are resistant to HIV infection. The CCR5 creates a receptor on the
surface of the white blood cells and that's what HIV binds too. People
who have a natural mutation to this gene prevent HIV from binding,
mutation does not lead to any other symptoms. The zinc finger protein is
used to take an HIV infected person and cleave their DNA and allow it
