Lecture 29:

Question 1

Gene Knockouts in Yeast Adv and DiAdv

Answer 1

ADV:
- Yeast can’t take up DNA from outside
- DNA introduced in chromosome

Question 2

Functional Genomics

Answer 2

Knockdown and analyze if they are lethal

Question 3

CRISPR acronym

Answer 3

clustered regulatory interspaced short palindromic repeats

Question 4

Cas

Answer 4

(CRISPR associated)

Flanks Cas

Question 5

CRISPR Cas9 Mech

Answer 5

Cas9 is directed to the target gene by guideRNAbase pairing.
Cas9 nuclease cleaves both strands of the target DNA producing a double-strand break.
The double-strand break can be repaired in two ways, either just sticking it back together (nonhomologous end-joining) or by homology-directed repair.

Question 6

DSB repair Types

Answer 6

Non-homologous end joining:
- Random bases often causing frameshift
Homology-directed repair:
- Requires expression of HDR template

Question 7

CRISPRi

Answer 7

inactive Cas9 so no cleavage just blockage

Question 8

CRISPRa

Answer 8

Cas9 fused to activation domain and thus promotes transcription id target sequence is upstream from gene of interest

Question 9

Cre-Lox system

Answer 9

This enables study of the function of a gene that is essential for viability, by knocking it out only in a tissue that is not essential (at least in the lab).

Cre interacts with loxP and results in alternative recombination of the gene

Question 10

CASGEVY good and bad

Answer 10

GOOD:
- alternative to stem cell that is high risk
- It is a one time treatment

BAD:
- Off-target effects guide RNA base pairs somewhere else
- Societal equity