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1. How can scientists use the CRISPR/Cas9 system used to mutate a gene? (Choose ALL correct answers)

[] Cas9 and the RNA it binds to can be put into cells by scientists in order to induce breaks at specific DNA sequences
[] once the DNA is cut, repair mechanisms of the cell can make changes to the DNA at that specific site
[] Cas9 binds with the transcribed mRNA from the gene and interferes with translation so that the protein is never produced
[] Cas9 can bind to the promoter of the gene to change the frequency of gene transcription

2. Which of the following are features of the CRISPR/Cas9 system? (Choose ALL correct answers)

[] Cas9 uses RNA to find matching sequences of DNA
[] Cas9 makes double-stranded breaks in specific sequences of DNA
[] Cas9 makes random double-stranded breaks in DNA
[] Cas9 makes breaks in RNA that are targeted and specific

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Bunny Greenfelder
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5 Oct 2018
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Related questions

Read the summary and answer questions in bold below:

In this post, we’ll be focusing on Tyler Jacks and his graduate student Francisco J. Sánchez-Rivera’s review, “Applications of the CRISPR-Cas9 system in cancer biology,” circa 2015 in Nature Reviews. So we understand the context of the authors, Jacks is the director of the Koch Institute for Integrative Cancer Research at MIT. His lab has a large mouse experimental component (MIT 2017). This sets the stage for the angle we’ll be reading from. In all, it’s a nice roundup of his and colleagues' cancer applications of CRISPR-Cas9 with great graphics, but leaves a bit desired in crossing the bridge from mouse to human.

Briefly, the CRISPR-Cas9 system stems from the prokaryotic immune system, where the RNA-guided DNA endonuclease Cas9 uses a single guide RNA (sgRNA) containing a CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) that binds Cas9 and directs it to the genomic sequence of interest, adjacent to a photospacer adjacent motif (PAM). Cas9 causes a precisely-directed double-stranded break (DSB) in the DNA, that gets repaired via non-homologous end joining (NHEJ) or homology-directed repair (HDR).

With NHEJ, this can disrupt the gene by creating Indels: frameshifts and stop codons. In cancer, individual oncogenes or tumor suppressors can be targeted, or drug response modulators, or synthetic lethality genes. With a multiplexed knockout approach, combinatorial vulnerabilities and epistatic relationships can be assessed.

For gene modification via HDR, target single or double-stranded DNA can be introduced, resulting in precise gene knock-ins, with potential effects of introducing point mutations to mimic cancers, fluorescent reporters/synthetic tags can be created, conditional (Cre-lox/Flp-FRT) constructs can used to turn off/on a mutation, non-coding elements like enhancers/insulators/promoters can turn on proto-oncogenes, and ultimately, HDR can be used for gene correction.

With Cas9 and 2 sgRNAs, there can be genomic rearrangements, leading to deletions, inversions (like EML4-ALK), and translocations (like BCR-ABL).

Dead Cas9 (dCas), which lacks its endonuclease activity via mutation to its HNH and RuvC domains, can be used for reversible, transcriptional repression or activation of coding and non-coding genes, or chromatin modification, via an effector.

Much of the practical applications of CRISPR-Cas9 is in ES cells and mouse models, both germinal and somatic (GEMMs and nGEMMs). With this technology, a plethora of GEMM/nGEMM models of cancer can be created, some with the ability to turn on/off the Cas9 system, and multiplexed sgRNAs can help gain further understanding of multi-hit cancers. Patient derived xenografts (PDX) mice can be assayed with CRISPR-Cas9 to determine different treatment efficacies. Entire libraries of sgRNA can be screened in cancer cell lines for enrichment/depletion with Cas9, and dCas9-activators/repressors can be used for complementarity, both of which providing insights into drug targets, synthetic lethality, and chemotherapy modulators.

The delivery mechanisms for the system are diverse, and include plasmids, lentivirus, adenovirus, nanoparticles, hydrodynamic gene transfer, and protein-based. The authors also cite integrating Cas9 into the GEMMs, so it’s one less thing needed in the delivery vehicle. This of course would not be applicable to human.

Ultimately, human cancer treatment is the goal. Yes, we know the boundaries between ES/mice experimental models and human embryos have already been broken, with an international team targeting the autosomal dominant mutation in MYBPC3, which causes cardiac myopathy (Ledford 2017). But the current compromise position of CRISPR-Cas9 technology toward therapeutics and treatment, beyond basic science understanding, is using primary cells, organoids, PDX, or personalized GEMMs to create genetic and drug screens.

In theory, what would it take to bring CRISPR-Cas9 to the ultimate, targeted gene therapy via either NHEJ or HDR, in human cancer? Are there cancers that are better bets for CRISPR-Cas9 than others, delivery vehicles that are safer or better optimized? Is CRISPR-Cas9 really “on target” enough to introduce into a person, and how do we best assess?

I. Signaling cascades (4 pts)

1. Which of the following is an example of a signaling cascade?

A. Binding of lactose to the lac repressor

B. Binding of the lac repressor to the lac operon

C. The series of proteolytic digests that occur during clotting

D. The Collins Toggle Switch

2. Why is it important that signaling cascades have negative feedback loops?

II. Metabolism (4 pts)

1. What is a benefit of a negative feedback allosteric regulator?

A. It prevents starting materials from being unnecessarily consumed.

B. It regulated metabolism without requiring new protein synthesis.

C. It can be rapidly reversed as conditions change.

D. All of the above.

2. How is a metabolic cycle, like the citric cycle, distinct from a metabolic pathway, like glycolysis?

III. Transcription (4 pts)

1. Transcription factors, repressors, and activators are examples of proteins that _________.

A. randomly activate RNA polymerases.

B. bind specific DNA sequences.

C. bind specific rRNA sequences.

D. are found in the lysosome.

2. The trp operon is attenuated by a stem-loop structure that can form near RNA polymerase. The RNA sequencing encoding the stem loop is:

5’-AUACCCAGCCCGCCUAAUGAGCGGGCUU-3’

The stem loop is composed of 7 nucleotides which perfectly base pair. Indicate the two halves of the stem loop on the sequence above.

IV. DNA replication/repair/genome editing (4 pts)

1. What is a consequence of the need for RNA primers during DNA replication?

A. Linear chromosomes gradually shorten each cell division.

B. Transcription factors often trigger DNA replication.

C. RNA primers are not actually needed for DNA replication.

D. Lagging strands can be avoided.

2. CRISPR-mediated genome editing has gene-specific components: an sgRNA, and a double-stranded repair template. Describe the role of the sgRNA in directing genome editing.

V. Genetic circuits (4 pts)

1. Which statement best describes the mechanism of the toggle switch?

A. Inducers must be continuously applied to maintain one of two states.

B. Competing repressors keep the cell in one of two transcriptional states.

C. An activator and a repressor keep the cell in one of two states.

D. A pair of repressors cause the cell to oscillate between two states without input.

2. How is bistability achieved by the Collins toggle switch?

Which of the following did Erwin Chargaff observe?

A)

Base composition changes as the organism ages.

B)

Base composition of DNA does not vary from one species toanother.

C)

In all species the number of adenines equals the number ofguanines.

D)

In all species A + T = G + C.

E)

DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)

the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)

the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)

the temperature at which double-stranded DNA is completelymelted.

D)

the temperature at which double-stranded DNA has become 50%single-stranded.

E)

the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)

Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)

Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)

Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)

A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)

Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)

Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)

Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)

When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)

None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)

DNA ligase

B)

Restriction endonuclease

C)

Reverse transcriptase

D)

DNA polymerase I

E)

Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)

Reverse transcriptase

B)

Alkaline phosphatase

C)

DNA ligase

D)

DNA polymerase I

E)

Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)

Alkaline phosphatase

B)

Reverse transcriptase

C)

Restriction endonuclease

D)

DNA ligase

E)

DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)

An origin of replication

B)

A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)

Unique restriction sites for insertion of DNA.

D)

Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)

All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)

Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)

cDNA libraries contain cDNAs made from mRNA.

C)

Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)

Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)

cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

A)

dNTPs

B)

DNA containing the sequences to be amplified

C)

DNA ligase to connect the fragments together

D)

Primers complementary to each end of the sequence to beamplified

E)

Heat stable Taq polymerase

How can a fusion protein be formed?

A)

Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)

Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)

Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)

Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)

A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)

The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)

The reaction is only dependent on the presence of GFP and oneother cofactor.

C)

It is easily cloned and expressed in bacterial cells.

D)

The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)

It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)

Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)

Create a fusion with green fluorescent protein (GFP).

C)

Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)

A and B might work.

E)

A, B, and C might work.

F)

None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)

The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)

A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)

Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)

Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)

The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

A)

3 Gm

B)

300 Kb

C)

3 Mb

D)

3 Gb

E)

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)

Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)

Eukaryotic genes often encode introns that are spliced outbefore translation.

C)

Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)

Eukaryotic genes are typically arranged in operons.

E)

Prokaryotic genes often encode exons that are spliced out beforetranslation

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