Lecture 9 - Genetic Medicine

Question 1

can be used to remove, change, and add DNA after cutting it

Answer 1

CRISPR/Cas9

Question 2

early models of genome editing performed the same functions as current ones, but the largest difference is:

Answer 2

the newer ones (like CRISPR-Cas9) are easier to program and more efficient

Question 3

when was the CRISPR sequence discovered and how?

Answer 3

1987, discovered that the bacteriophage genome had strage repeat sequences but at the time their function was unknown

Question 4

in 2009, it was found that the repeat CRISPR sequences function in:

Answer 4

protecting against viruses

Question 5

in 2012, it was found that Cas9:

Answer 5

works alongside CRISPR to target and cut specific DNA systems

Question 6

what does CRISPR-Cas9 stand for?

Answer 6

• CRISPR = clustered regularly interspaced short palindromic repeats
• Cas9 = CRISPR associated protein 9

Question 7

CRISPR-Cas9 is part of the prokaryotic immune system that:

Answer 7

defends against viruses

Question 8

what is the purpose of guide RNA?

Answer 8

binds to virus sequences

Question 9

how does the guide RNA/Cas9 complex work?

Answer 9

binds to invading DNA and cuts it up

Question 10

Cas9 is recruited to the DNA target site by the duplex:

Answer 10

tracrRNA:crRNA (guide RNA)

Question 11

binds the complementary DNA upstream of the PAM sequence

Answer 11

crRNA

Question 12

what is the PAM sequence?

Answer 12

protospacer adjacent motif

Question 13

what are the three key elements to the CRISPR-Cas9 system?

Answer 13

1) the Cas9 DNA endonuclease
2) crRNA
3) a trans-activating crRNA (tracrRNA)

Question 14

contains a 20bp sequence complementary to the target

Answer 14

crRNA

Question 15

acts as a bridge between the crRNA and the Cas9 system

Answer 15

trans-activating crRNA (tracrRNA)

Question 16

DNA endonuclease is targeted to a DNA sequence via a ______, upstream of the ______, resulting in a ______ upstream of the NGG

Answer 16

a single guide RNA (sgRNA) sequence, protospacer-associated motif (PAM), bp double-strand break (DSB)

Question 17

using a CRISPR-Cas9 system, the resulting double-strand breaks are sealed back together by either ____ or ____

Answer 17

non-homologous end joining (NHEJ), homology-directed repair (HDR)

Question 18

what is a challenge to using CRISPR-Cas9?

Answer 18

off-target DNA cleavage

Question 19

the 3’ end of the DNA target sequence must have a:

Answer 19

proto-spacer adjacent motif (PAM) sequence (5’-NGG-3’)

Question 20

where does the Cas9 nuclease cleave the DNA relative to the PAM sequence?

Answer 20

approximately three bases upsteam of the PAM

Question 21

the PAM sequence itself is absolutely required for cleavage, but is NOT part of:

Answer 21

the sgRNA sequence

Question 22

how can Cas9 be delivered?

Answer 22

as a DNA or mRNA molecule encoding for the Cas9 gene, or it may be delivered as a functional ribonucleoprotein (RNP)

Question 23

what is the challenge with delivering Cas9?

Answer 23

delivering the cargo across the cell membrane

Question 24

what are the three elements required for base editing?

Answer 24

1) a Cas nickase or Cas fused to a deaminase that makes the edit
2) a gRNA targeting Cas to a specific locus
3) a target base for editing within the the editing window specified by the Cas protein

Question 25

what does base editing do?

Answer 25

changes a single base pair in the DNA sequence

Question 26

uses an engineered reverse transcriptase fused to Cas nickase and a prime-editing guide RNA to change a base pair in the DNA sequence

Answer 26

prime-editors

Question 27

catalytically inactive, or "dead", Cas9 (dCas9), in which point mutations evade DNA cleavage by the HNH or RuvC domains, can be repurposed as a _____ or _____

Answer 27

transcription activator (CRISPRa), transcription repressor (CRISPRi)

Question 28

what does CRISPRa do?

Answer 28

a transcription activator which increases gene expression

Question 29

what does CRISPRi do?

Answer 29

a transcription repressor which knocks down gene expression

Question 30

the dCas9-p300 system is used for:

Answer 30

epigenetic activation

Question 31

dCas9 fused to the core catalytic domain of p300 can:

Answer 31

acetylate target sites in the genome

Question 32

in the dCas9-p300 system, acetylation of the target gene synergizes with the action of:

Answer 32

transcription factors and RNA polymerase II, resulting in transcriptional upregulation

Question 33

CRISPR is capable of making highly specific, permanent _______ that are more likely to reduce _______

Answer 33

genetic modifications, target gene function

Question 34

CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including:

Answer 34

resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis

Question 35

go review slide 268

Answer 35

you got this babe

Question 36

the single cut CRISPR strategy restores the ______ protein

Answer 36

dystrophin

Question 37

diseases of the eye and ear are being treated by CRISPR-Cas9 ____ in clinical trials

Answer 37

in vivo (directly in the living eye/ear)

Question 38

type of CRISPR-Cas9 therapy which is useful for blood diseases like sickle cell anemia, hemophilia, and CAR T cancer

Answer 38

ex vivo therapy

Question 39

what are somatic cells?

Answer 39

any cell in a living organism other than a reproductive cell

Question 40

what are germline cells?

Answer 40

cells segregated and involved in the reproductive process that are passed onto the progeny

Question 41

what are some ethical and legal concerns when it comes to using CRISPR-Cas9?

Answer 41

- safety: if gene editing specificity is imperfect, should we still use it? - should human gene editing be allowed? - should parents be allowed to edit the genomes of their unborn children