Genetics Lab #3 CRISPR-Cas9

What are the Functions of the Cas9 Protein?

A DNA binding protein A RNA binding protein A helicase that unwinds DNA A nuclease

What is CRISPR-Cas9?

A system that allows precise genome editing by cutting out DNA and inserting new DNA in its place.

How are the zinc finger nuclease (ZFN) system of gene-editing and the CRISPR-Cas9 system similar?

Both cause a cut (double-stranded break) in the target DNA

Choose all sequences that would enable gene editing at a specific gene locus

CCT AGG GGG CCC

Not only bacteria but also humans employ CRISPR as the primary adaptive immune mechanism TRUE or FALSE

FALSE

The CRISPR system can provide immunity against bacteriophages with RNA genomes: True or False

FALSE

Deactivating both Cas9 nuclease domains would prevent Cas9 from binding to its target DNA

FALSE Binding would be possible, however cleavage would be inactivated

When carrying out gene editing with CRISPR-Cas9, HDR is always preferred because it is more precise.

FALSE During CRISPR-Cas9, it is possible to re-cut repaired sequences, especially since targeted sequences will accrue indels over a day or two. As a result, scientists can rely on NHEJ to achieve high frequencies of knockouts in many cell types.

Cystic fibrosis is a disorder caused by deletion of 3 nucleotides in the CFTR gene. You are planning to fix this mutation using CRISPR-Cas9 technology. Which repair pathway will you use?

HDR

To correct a gene using CRISPR-Cas9 gene editing, which of the following will you select:

HDR inhibitors HDR is not desirable as a repair mechanism with CRISPR-Cas9

Your colleague is using the nickase version of Cas9 (that only cuts one DNA strand instead of two) to modify the genomic DNA. Which one of the cellular repair pathways is she planning to use?

Homologous recombination

You design a CRISPR-Cas9 gene editing strategy which targets the DNA region coding for its own gRNA. Select all the correct statements

If the gRNA acquires mutation, it will still be able to target itself PAM is located in the gRNA coding region.

What are Spacer Sequences in Bacterial CRISPR?

Incorporated into the CRISPR array Complementary to a viral genome

NHEJ can result in. . .

Insertions Deletions No changes

What is the advantage of CRISPR-Cas9 compared to other gene-editing methods? Multiple answers are possible.

It can edit anywhere in the genome. It is easily programmable. CRISPR-Cas9 can bind and cut DNA, while other editors only bind. It is cheap and easy for researchers to use.

Why have CRISPR systems become such powerful tools?

It is easy and quick to target novel sequences due to the modular and programmable nature of RNA-DNA interactions

In sickle cell anemia, structural change of hemoglobin (the protein that carries oxygen in red blood cells) leads to abnormally sickle-shaped red blood cells that are prone to blocking capillaries and causing anemia. What is the reason that hemoglobin is different in patients with sickle cell?

Mutation of a single nucleotide that leads to a change of an amino acid in the protein

After creating a targeted double-stranded break in the genomic DNA of a cell using CRISPR-Cas9, you notice that the cell has used NHEJ for repair. As a result, some indels (insertions and deletions in DNA) have been introduced in the region, which do not allow for efficient base pairing between gRNA and target DNA. Will you be able to cut the target location in this cell again with the same gRNA?

No A new gRNA sequence complementary to the targeted DNA will be required

Which statement is NOT correct about zinc finger nucleases? -they contain zinc finger domains that tightly bind to zinc ions. -Zinc finger domains can function independently from the rest of the protein for binding to specific DNA sequences. -Precision of DNA sequence recognition decreases by increasing the number of sequence specific zinc finger domains. -Engineering zinc finger nucleases that recognize any desired DNA sequence with high precision remains extremely labor-intensive

Precision of DNA sequence recognition decreases by increasing the number of sequence specific zinc finger domains

Which of the following are necessary for the CRISPR gene editing system to function? Multiple answers are possible.

Recognition of the PAM sequence Formation of the Cas9-gRNA complex Binding of gRNA to its complementary DNA strand

potential applications for CRISPR-Cas9 editing

Research tool to elucidate biological mechanisms Treatment of human disease in the embryonic stage of development Agricultural Crop engineering Elimination of vector-borne diseases

If you wanted to generate a variant of Cas9 that can bind but not cut both strands of template DNA, which domains would you mutate?

RuvC Domain HNH Domain RuvC domain responsible for cleavage of the displaced noncomplementary (non-target) DNA strand HNH domain cleaves the complementary (target) strand of the DNA target

If you wanted to generate a variant of Cas9 that cuts only one strand of template DNA, creating a "nickase", which domains would you mutate?

RuvC domain HNH domain

Cas9 contains two nuclease domains, and each cuts only one strand of target DNA. By deactivating one of the nucleases, we can create a Cas9 that creates nicks (single-stranded breaks) instead of double-stranded breaks.

TRUE

NHEJ is more common and more error prone than HDR

TRUE

When a double-stranded DNA break occurs, the HDR and NHEJ pathways are in competition with one another to repair the break.

TRUE

Based on what you learned about the CRISPR system, what is the most straightforward way to ensure that the spacer sequence within the bacterium's own genome is not cut by the CRISPR system?

The spacer sequence in the bacterial genome does not have an adjacent PAM sequence

NHEJ is more common to repair DNA than HDR. How could you shift the balance towards HDR? More than one answer can be selected.

Use protein inhibitors of NHEJ proteins. Deliver the Cas9-gRNA complex alongside a huge surplus of donor templates.

How Long Does the Programmable Part of a gRNA Need to Be?

We need sequences longer than 20 base pairs (bp) to uniquely identify a sequence in the human genome. Therefore, when working in human cells, we use gRNAs with 30 bp complementary to target DNA.-FALSE

what is the native biological role of CRISPR?

a defense system against invading viruses a bacterial adaptive immune response

Homology-directed repair is:

is far less error-prone than Non-Homologous End Joining and can be used by researchers to introduce specific nucleotide changes in the target sequence. uses a template strand to fix double stranded breaks in DNA. uses an excess of synthetic DNA to serve as a custom made HDR donor template to outcompete homologous DNA from the second allele.

Non-Homologous End Joining (NHEJ) repair ...

is often error-prone and can lead to gene inactivation by disrupting the sequence.

All of the following factors are necessary for the CRISPR system to provide immunity against a bacteriophage except: -the spacer sequence that keeps the memory of the bacteriophage. -the crRNA complementary to the DNA of the bacteriophage. -the Cas nuclease. -specific sequences in the genome of the bacteriophage.

specific sequences in the genome of the bacteriophage.