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13th Edition
William S Klug, Michael A Palladino , Darrell Killian
All Chapters Solutions Included - Ch 1 to 26 ✅
Special Topics 1 to 6 Included ✅
,Special Topic 1: CRISPR-Cas and Genome Editing
Review Question Answers
1. Innate immunity describes cellular defense mechanisms that are not universal and not targeted to a
specific pathogen. By contrast, adaptive immunity describes an evolving mechanism whereby exposure
to a pathogen results in improved defense upon subsequent exposure to that same pathogen.
2. Bacteria that became resistant to a given phage strain acquired new spacer sequences in their CRISPR
loci that matched portions of the phage genome. Deletion or mutation of these spacers is correlated with
renewed sensitivity to the phage, and experimental insertion of these sequences into the CRISPR loci of
sensitive bacteria made them resistant.
3. The three steps are acquisition, crRNA biogenesis, and interference. First, CRISPR-associated (Cas)
proteins cleave viral DNA into smaller fragments, which insert proximal to the CRISPR leader sequence
(acquisition of new spacer). Next, transcription of the CRISPR locus yields a long transcript, which is
processed into short CRISPR-derived RNAs (crRNA biogenesis). These crRNAs consist of a single spacer
flanked by repeat sequences. Finally, mature crRNAs associate with a Cas nuclease or nuclease complex,
recruiting them to complementary sequences of phage DNA, which they cleave (interference).
4. The type II CRISPR-Cas9 system of S. pyogenes was selected due to its simplicity. Only a few components
are needed, chief among them being the Cas9 nuclease, which plays a role in all three steps of CRISPR-
Cas-mediated defense.
5. The Cas9 nuclease distinguishes between “self” and “non-self” (foreign) DNA by the absence or presence,
respectively, of a PAM (protospacer adjacent motif) sequence. During spacer acquisition, the Cas9
nuclease cleaves invading DNA adjacent to (but not including) a PAM. During CRISPR-Cas-mediated
defense, Cas9 will cleave the DNA only if it is adjacent to a PAM sequence (and, therefore, “non-self” or
foreign).
6. A single guide RNA (sgRNA) is an engineered RNA molecule that takes the place of the crRNA/tracrRNA
duplex. The sgRNA contains 20 nucleotides of a crRNA sequence linked to the minimal functional
sequence of the tracrRNA.
7. Repair of CRISPR-Cas9 editing by NHEJ, an error prone pathway, would likely disrupt the function of the
target gene. This would be appropriate if the goal were to inactivate a harmful dominant allele in a
heterozygous cell. Repair by HDR, when a suitable donor template is provided, allows precise
substitutions as well as additions or deletions. This type of repair would be appropriate if the goal were
to correct a homozygous recessive mutation.
8. Mutations in the HNH and RuvC domains of Cas9, known as “dead” Cas9 (dCas9) eliminate its nuclease
activity. However, dCas9 can still bind DNA in a programmable manner based on complementary base-
pairing of the sgRNA and the target DNA sequence. Scientists have fused dCas9 to other proteins to
develop other tools. By attaching a transcriptional activation domain or repression domain to Cas9, it can
be used to activate or repress transcription of target genes. Histone-modifying enzymes, such as histone
acetyl transferase or DNA methyltransferase can also be attached to dCas9 to regulate chromatin
packaging. A fluorescent protein attached to dCas9 can be used to fluorescently label target sequences.
9. There are multiple examples mentioned in text. One example is the modification of the pig genome to
make pigs resistant to the porcine respiratory and reproductive syndrome virus, which negatively impacts
the pig farming industry. Another example is the editing of the pig genome to make pig organs safe and
immunologically compatible for xenotransplantation into humans. Other examples include engineering
, drought tolerance into crops and gene drives that may be used to reduce or eradicate invasive species or
species that serve as disease vectors.
10. For genetic conditions caused by a dominant mutation, a strategy that disrupts the dominant allele in a
heterozygote using an allele-specific sgRNA would be appropriate. The resulting loss-of-function
mutation would allow expression of the recessive wild-type allele and restoration of typical gene
function. For recessive genetic conditions, a strategy that uses a donor template to correct the mutation
in the disease-causing allele is often necessary to restore wild-type gene function.
Discussion Question Answers
1. The use of ZFNs and TALENs was slow and technically challenging, since they required the engineering
of proteins that could recognize and cut a specific DNA sequence. By contrast, the specificity of the
CRISPR-Cas system requires only a sgRNA that is complementary to the target sequence and the
presence of a PAM in the target.
2. A specific 20-nucleotide sequence occurs once every 4
20
or 1.1 × 1012 base pairs. A PAM ′ ′
(5 -NGG-3 )
occurs once every 16 base pairs in a random sequence. Both will occur once every 1.76 × 1013 base pairs
(remember to multiply the individual probabilities to calculate the probability of both occurring at the
same time). Given that the haploid human genome is 3.2 × 10
9
, finding both by pure chance would be
bp
highly improbable.
3. Safety concerns would primarily center on the specificity of the edits, given the possibility of off-target
edits and unwanted genomic changes. Efficiency of editing is also a concern—a mutant gene might be
edited in some cells but not in others, raising the possibility that the disease state may persist. This is also
an ethical concern—should this treatment be attempted if there is a low chance of success? Making
potentially heritable changes to embryos is another ethical concern. The question of whether CRISPR-
Cas editing should be used to engineer desirable traits is itself an ethical question.
4. Both systems rely on complementary base pairing to recruit a protein to target sequences. In both
systems, the guide molecule is a short RNA molecule that is derived from a larger transcript. The systems
differ in their targets—mRNA for miRNA/RISC vs. genomic DNA for CRISPR-Cas. While the
miRNA/RISC system is limited to gene silencing, editing by CRISPR-Cas can result in silencing (by gene
disruption), removal of a single protein domain (by targeted deletion), or the substitution of a functional
gene for a mutant gene.
5. Cas9 and Cas12a are both Cas nucleases capable of making double-stranded breaks in the DNA.
However, they differ in a few key ways. Cas9 requires a crRNA and a tracrRNA whereas Cas12a only
requires a crRNA. Cas9 uses the PAM sequence ′
5 -NGG-3
′
whereas Cas12a uses the PAM sequence
′
5 -TTTV-3
′
(V is anything other than T). This means that Cas9 is better for targeting G/C-rich DNA and
Cas12a is better for targeting A/T-rich DNA. Cas9 and Cas12a also differ in how they cut target DNA;
Cas9 produces predominantly blunt-ended DNA fragments while Cas12a produces staggered cuts in the
DNA. One is not better than the other. Rather, for any given genome-editing goal, one may have
advantages over the other.
6. Both cystic fibrosis and hemophilia would be good candidates for gene editing by CRISPR-Cas, since
each is caused by a recessive mutation of a single gene. Depending on the specific type of mutation
involved, either editing or substitution of a wild-type allele would correct the problem. Down syndrome
would be a poor candidate for treatment using CRISPR-Cas because the condition is caused by an extra
copy of an entire chromosome (trisomy 21) or by translocation of a critical portion of chromosome 21.
This cannot be corrected by gene editing. Brain cancer is also a poor candidate, since its cause is complex
and not fully understood.
7. For both adult somatic cells and embryos, off-target edits could result in unexpected deleterious
phenotypes. The risk could be evaluated and potentially avoided in somatic cell editing. In embryos,
, however, these unexpected phenotypes could affect development and/or viability and, should the treated
embryo survive and grow to adulthood, these unintended edits could be heritable.
!"#$%&'( ") *%#%'+$(
13th Edition
William S Klug, Michael A Palladino , Darrell Killian
All Chapters Solutions Included - Ch 1 to 26 ✅
Special Topics 1 to 6 Included ✅
,Special Topic 1: CRISPR-Cas and Genome Editing
Review Question Answers
1. Innate immunity describes cellular defense mechanisms that are not universal and not targeted to a
specific pathogen. By contrast, adaptive immunity describes an evolving mechanism whereby exposure
to a pathogen results in improved defense upon subsequent exposure to that same pathogen.
2. Bacteria that became resistant to a given phage strain acquired new spacer sequences in their CRISPR
loci that matched portions of the phage genome. Deletion or mutation of these spacers is correlated with
renewed sensitivity to the phage, and experimental insertion of these sequences into the CRISPR loci of
sensitive bacteria made them resistant.
3. The three steps are acquisition, crRNA biogenesis, and interference. First, CRISPR-associated (Cas)
proteins cleave viral DNA into smaller fragments, which insert proximal to the CRISPR leader sequence
(acquisition of new spacer). Next, transcription of the CRISPR locus yields a long transcript, which is
processed into short CRISPR-derived RNAs (crRNA biogenesis). These crRNAs consist of a single spacer
flanked by repeat sequences. Finally, mature crRNAs associate with a Cas nuclease or nuclease complex,
recruiting them to complementary sequences of phage DNA, which they cleave (interference).
4. The type II CRISPR-Cas9 system of S. pyogenes was selected due to its simplicity. Only a few components
are needed, chief among them being the Cas9 nuclease, which plays a role in all three steps of CRISPR-
Cas-mediated defense.
5. The Cas9 nuclease distinguishes between “self” and “non-self” (foreign) DNA by the absence or presence,
respectively, of a PAM (protospacer adjacent motif) sequence. During spacer acquisition, the Cas9
nuclease cleaves invading DNA adjacent to (but not including) a PAM. During CRISPR-Cas-mediated
defense, Cas9 will cleave the DNA only if it is adjacent to a PAM sequence (and, therefore, “non-self” or
foreign).
6. A single guide RNA (sgRNA) is an engineered RNA molecule that takes the place of the crRNA/tracrRNA
duplex. The sgRNA contains 20 nucleotides of a crRNA sequence linked to the minimal functional
sequence of the tracrRNA.
7. Repair of CRISPR-Cas9 editing by NHEJ, an error prone pathway, would likely disrupt the function of the
target gene. This would be appropriate if the goal were to inactivate a harmful dominant allele in a
heterozygous cell. Repair by HDR, when a suitable donor template is provided, allows precise
substitutions as well as additions or deletions. This type of repair would be appropriate if the goal were
to correct a homozygous recessive mutation.
8. Mutations in the HNH and RuvC domains of Cas9, known as “dead” Cas9 (dCas9) eliminate its nuclease
activity. However, dCas9 can still bind DNA in a programmable manner based on complementary base-
pairing of the sgRNA and the target DNA sequence. Scientists have fused dCas9 to other proteins to
develop other tools. By attaching a transcriptional activation domain or repression domain to Cas9, it can
be used to activate or repress transcription of target genes. Histone-modifying enzymes, such as histone
acetyl transferase or DNA methyltransferase can also be attached to dCas9 to regulate chromatin
packaging. A fluorescent protein attached to dCas9 can be used to fluorescently label target sequences.
9. There are multiple examples mentioned in text. One example is the modification of the pig genome to
make pigs resistant to the porcine respiratory and reproductive syndrome virus, which negatively impacts
the pig farming industry. Another example is the editing of the pig genome to make pig organs safe and
immunologically compatible for xenotransplantation into humans. Other examples include engineering
, drought tolerance into crops and gene drives that may be used to reduce or eradicate invasive species or
species that serve as disease vectors.
10. For genetic conditions caused by a dominant mutation, a strategy that disrupts the dominant allele in a
heterozygote using an allele-specific sgRNA would be appropriate. The resulting loss-of-function
mutation would allow expression of the recessive wild-type allele and restoration of typical gene
function. For recessive genetic conditions, a strategy that uses a donor template to correct the mutation
in the disease-causing allele is often necessary to restore wild-type gene function.
Discussion Question Answers
1. The use of ZFNs and TALENs was slow and technically challenging, since they required the engineering
of proteins that could recognize and cut a specific DNA sequence. By contrast, the specificity of the
CRISPR-Cas system requires only a sgRNA that is complementary to the target sequence and the
presence of a PAM in the target.
2. A specific 20-nucleotide sequence occurs once every 4
20
or 1.1 × 1012 base pairs. A PAM ′ ′
(5 -NGG-3 )
occurs once every 16 base pairs in a random sequence. Both will occur once every 1.76 × 1013 base pairs
(remember to multiply the individual probabilities to calculate the probability of both occurring at the
same time). Given that the haploid human genome is 3.2 × 10
9
, finding both by pure chance would be
bp
highly improbable.
3. Safety concerns would primarily center on the specificity of the edits, given the possibility of off-target
edits and unwanted genomic changes. Efficiency of editing is also a concern—a mutant gene might be
edited in some cells but not in others, raising the possibility that the disease state may persist. This is also
an ethical concern—should this treatment be attempted if there is a low chance of success? Making
potentially heritable changes to embryos is another ethical concern. The question of whether CRISPR-
Cas editing should be used to engineer desirable traits is itself an ethical question.
4. Both systems rely on complementary base pairing to recruit a protein to target sequences. In both
systems, the guide molecule is a short RNA molecule that is derived from a larger transcript. The systems
differ in their targets—mRNA for miRNA/RISC vs. genomic DNA for CRISPR-Cas. While the
miRNA/RISC system is limited to gene silencing, editing by CRISPR-Cas can result in silencing (by gene
disruption), removal of a single protein domain (by targeted deletion), or the substitution of a functional
gene for a mutant gene.
5. Cas9 and Cas12a are both Cas nucleases capable of making double-stranded breaks in the DNA.
However, they differ in a few key ways. Cas9 requires a crRNA and a tracrRNA whereas Cas12a only
requires a crRNA. Cas9 uses the PAM sequence ′
5 -NGG-3
′
whereas Cas12a uses the PAM sequence
′
5 -TTTV-3
′
(V is anything other than T). This means that Cas9 is better for targeting G/C-rich DNA and
Cas12a is better for targeting A/T-rich DNA. Cas9 and Cas12a also differ in how they cut target DNA;
Cas9 produces predominantly blunt-ended DNA fragments while Cas12a produces staggered cuts in the
DNA. One is not better than the other. Rather, for any given genome-editing goal, one may have
advantages over the other.
6. Both cystic fibrosis and hemophilia would be good candidates for gene editing by CRISPR-Cas, since
each is caused by a recessive mutation of a single gene. Depending on the specific type of mutation
involved, either editing or substitution of a wild-type allele would correct the problem. Down syndrome
would be a poor candidate for treatment using CRISPR-Cas because the condition is caused by an extra
copy of an entire chromosome (trisomy 21) or by translocation of a critical portion of chromosome 21.
This cannot be corrected by gene editing. Brain cancer is also a poor candidate, since its cause is complex
and not fully understood.
7. For both adult somatic cells and embryos, off-target edits could result in unexpected deleterious
phenotypes. The risk could be evaluated and potentially avoided in somatic cell editing. In embryos,
, however, these unexpected phenotypes could affect development and/or viability and, should the treated
embryo survive and grow to adulthood, these unintended edits could be heritable.
