Chapter 7: 7.2 Gene Editing and CRISPR Flashcards
What methods are there to edit the genome?
- Zinc Finger Nucleases (ZFNs)
- Transcription Activator-Like Effector Nucleases (TALENs)
- CRISPR-Cas9
ZFNs:
What do ZFNs contain?
- Zinc finger (DNA-binding) domain
- DNA cleavage domain
ZFNs:
How many zinc fingers are engineered typically? What do they recognize?
- 3-9 zinc fingers
- Recognize a 3 base pair sequence
ZFNs:
Characteristics of the DNA cleavage domain
- A nonspecific nuclease (FokI)
- Must work as a dimer
TALENs:
What do TALENs contain?
- TAL effector DNA-binding domain
- DNA cleavage domain
TALENs:
What are characteristics of the TALE DNA binding domain?
- Has a conserved 33-34 amino acid region that has a variable 12th and 13th position
TALENs:
What is the function of the structure of the TALE DNA binding domain?
- Allows it to bind specifically to the DNA
True or False:
Each TALE DNA binding domain binds to 3 nucleotides
False, each TALE binds a signle nucleotide
TALENs:
Characteristics of the cleavage domain
- Has a nonspecific nuclease (FokI)
What does CRISPR stand for?
Clustered Regularly InterSpaced Palendromic Repeats
Where was CRISPR-Cas9 derived from?
Derived form bacterial defense mechanisms
CRISPR-Cas9:
What are characteristics of a single-guide RNA (sgRNA)?
Has a target sequence and a Cas9 interacting sequence
CRISPR-Cas9:
In the sgRNA, what precedes the target sequence?
PAM sequence
CRISPR-Cas9:
What does PAM stand for?
Protospacer adjacent motif
CRISPR-Cas9:
What is the function of the sgRNA?
Binds specifically to a region of the genome and Cas9 makes a double stranded break
In bacteria:
What is the purpose of CRISPR/Cas system?
A natural mechanism by which bacteria protect themselves against foreign DNA (e.g. phage DNA)
How does the CRISPR/Cas system function naturally in bacteria?
- Bacteria cleaves invading phage DNA, adds segments into the CRISPR array
- Transcription of the array contains mRNA with CRISPR repeats and invading DNA
- Repeat sequence binds to tracrRNA which provides a scaffold for Cas protein
- When the bacteria gets infected again with that phage, the complex base pairs with the phage DNA
- Cas cleaves the phage DNA
CRISPR-Cas9:
Cas9
Engineered version of the Cas endonuclease
CRISPR-Cas9:
Guide RNA
Engineered to bind to Cas9 and to a specific site on genomic DNA (site of choice)
How will DNA often repair itself after a double stranded break? What are the effects of this?
Non-Homologous End Joining (NHEJ)
* Can cause a deletion or frameshift which inactivates the gene
Genes can be introduced to a cell if it repairs itself through…
Homology Directed Repair (HDR)
Describe:
How genes can be inserted using HDR
- Add segments that match the sequences flanking the cleavage site
- When DNA is cleaved, HDR will insert donor DNA sequence into cleavage site during DNA repair
