Chapter 7: 7.2 Gene Editing and CRISPR

Question 1

What methods are there to edit the genome?

Answer 1

1. Zinc Finger Nucleases (ZFNs)
2. Transcription Activator-Like Effector Nucleases (TALENs)
3. CRISPR-Cas9

Question 2

ZFNs:

What do ZFNs contain?

Answer 2

1. Zinc finger (DNA-binding) domain
2. DNA cleavage domain

Question 3

ZFNs:

How many zinc fingers are engineered typically? What do they recognize?

Answer 3

• 3-9 zinc fingers
• Recognize a 3 base pair sequence

Question 4

ZFNs:

Characteristics of the DNA cleavage domain

Answer 4

• A nonspecific nuclease (FokI)
• Must work as a dimer

Question 5

TALENs:

What do TALENs contain?

Answer 5

1. TAL effector DNA-binding domain
2. DNA cleavage domain

Question 6

TALENs:

What are characteristics of the TALE DNA binding domain?

Answer 6

• Has a conserved 33-34 amino acid region that has a variable 12th and 13th position

Question 7

TALENs:

What is the function of the structure of the TALE DNA binding domain?

Answer 7

• Allows it to bind specifically to the DNA

Question 8

True or False:

Each TALE DNA binding domain binds to 3 nucleotides

Answer 8

False, each TALE binds a signle nucleotide

Question 9

TALENs:

Characteristics of the cleavage domain

Answer 9

• Has a nonspecific nuclease (FokI)

Question 10

What does CRISPR stand for?

Answer 10

Clustered Regularly InterSpaced Palendromic Repeats

Question 11

Where was CRISPR-Cas9 derived from?

Answer 11

Derived form bacterial defense mechanisms

Question 12

CRISPR-Cas9:

What are characteristics of a single-guide RNA (sgRNA)?

Answer 12

Has a target sequence and a Cas9 interacting sequence

Question 13

CRISPR-Cas9:

In the sgRNA, what precedes the target sequence?

Answer 13

PAM sequence

Question 14

CRISPR-Cas9:

What does PAM stand for?

Answer 14

Protospacer adjacent motif

Question 15

CRISPR-Cas9:

What is the function of the sgRNA?

Answer 15

Binds specifically to a region of the genome and Cas9 makes a double stranded break

Question 16

In bacteria:

What is the purpose of CRISPR/Cas system?

Answer 16

A natural mechanism by which bacteria protect themselves against foreign DNA (e.g. phage DNA)

Question 17

How does the CRISPR/Cas system function naturally in bacteria?

Answer 17

1. Bacteria cleaves invading phage DNA, adds segments into the CRISPR array
2. Transcription of the array contains mRNA with CRISPR repeats and invading DNA
3. Repeat sequence binds to tracrRNA which provides a scaffold for Cas protein
4. When the bacteria gets infected again with that phage, the complex base pairs with the phage DNA
5. Cas cleaves the phage DNA

Question 18

CRISPR-Cas9:

Cas9

Answer 18

Engineered version of the Cas endonuclease

Question 19

CRISPR-Cas9:

Guide RNA

Answer 19

Engineered to bind to Cas9 and to a specific site on genomic DNA (site of choice)

Question 20

How will DNA often repair itself after a double stranded break? What are the effects of this?

Answer 20

Non-Homologous End Joining (NHEJ)
* Can cause a deletion or frameshift which inactivates the gene

Question 21

Genes can be introduced to a cell if it repairs itself through…

Answer 21

Homology Directed Repair (HDR)

Question 22

Describe:

How genes can be inserted using HDR

Answer 22

1. Add segments that match the sequences flanking the cleavage site
2. When DNA is cleaved, HDR will insert donor DNA sequence into cleavage site during DNA repair