CRISPR

Question 1

What is genome-editing?

Answer 1

Cutting genomes using ‘DNA scissors’; CRISPR/Cas9; can be used to remove/add DNA to change the function.

Question 2

What are genomics?

Answer 2

The study of the entirety of an organism’s genes.

Question 3

What is Genome-wide association study (GWAS)?

Answer 3

A tool used in genetics research to associate specific genetic variations with particular diseases.

Question 4

What are functional genomics?

Answer 4

A study that describes gene functions and interactions.

Question 5

What are ZFNs?

Answer 5

Directed evolution.

Question 6

What are TALENs?

Answer 6

Clone new RVDs.

Question 7

When did CRISPR-Cas9 explode into popularity?

Answer 7

2012 onset.

Question 8

Who discovered the CRISPR sequence?

Answer 8

Yoshizumi Ishino

Question 9

What does CRISPR stand for?

Answer 9

Clustered Regularly Interspaced Palindromic Repeats

Question 10

When did the CRISPR win a Nobel prize for genome editing?

Answer 10

2020

Question 11

What is Cas9?

Answer 11

Protein part of the prokaryotic immune system that defends against viruses,.

Question 12

What recruits Cas9 to DNA target sites?

Answer 12

tracrRNA:crRNA duplex.

Question 13

What are the key elements of the CRISPR/Cas9 components?

Answer 13

1. Cas9 DNA endonuclease.
2. crRNA, which contains 20bp sequence complementary to target.
3. Trans-activating crRNA (tracrRNA) which acts as a bridge between crRNA and Cas9 enzyme.

Question 14

What is PAM?

Answer 14

Protospacer Adjacent motif (NGG)

Question 15

What is the mechanism of CRISPR/Cas9 DNA cleavage?

Answer 15

1. Dna endonuclease targets a DNA sequence via (sgRNA) single guide RNA sequence upstream of the PAM. This results in a 3bp ds break upstream of the NGG.
2. Resulting dsbs are repaired by either NHEJ or homology-directed repair (HDR).

Question 16

What are the off-target effects of CRISPR?

Answer 16

1. DNA bulge caused by 1-bp insertion.

2. RNA bulge caused by 1-bp deletion.

Question 17

What is the target sequence?

Answer 17

The 20 bp upstream the PAM.

Question 18

What is crRNA?

Answer 18

Crisper RNA; 17-20 nucleotide sequence to target DNA.

Question 19

Where does Cas9 cleave in the target DNA?

Answer 19

3 bases upstream the PAM.

Question 20

What is absolutely required for Cas9 cleavage?

Answer 20

The presence of a PAM.

Question 21

How may CRISPR be delivered? (form)

Answer 21

1. DNA/RNA molecule encoding the Cas9 gene.

2. Functional ribunucleoprotein.

Question 22

How may CRISPR cross the membrane?

Answer 22

1. Microinjection
2. Electroporatin
3. Chemical methods.

Question 23

What is the function of Cas nickase?

Answer 23

Makes edits in CRISPR to replace target DNA segments (specifed target base) SINGLE STRANDED CUT

Question 24

What is the function of gRNA?

Answer 24

Targets Cas to a specific locus.

Question 25

What are the components of the Base Editing complex?

Answer 25

Cas nickase, gRNA, target base.

Question 26

What components are involved in prime-editing?

Answer 26

Engineered reverse transcriptase fused to Cas9 nickase | Prime-editing RNA guide (pegRNA)

Question 27

What is the function of CRISPRa?

Answer 27

Used as transcription activators to increase gene expression.

Question 28

What is the function of CRISPRi?

Answer 28

Used as a transcription suppressor to decrease gene expression.

Question 29

What is the complex used in epigenome editing?

Answer 29

dCas9 with core catalytic domain of p300.

Question 30

What is the mechanism of epigenome editing?

Answer 30

Acetylation of target gene synergizes with activation of transcription factors and RNA Pol, resulting in upregulation of transcription.

Question 31

How is CRISPR used in screening?

Answer 31

1. Screens for novel genes that regulate known phenotypes. 2. Screen for drug resistance. 3. screen for tumor metastasis and cell viability.

Question 32

When is in vivo CRISPR editing useful?

Answer 32

Diseases of the eye and ear.

Question 33

When is ex vivo CRISPR editing useful?

Answer 33

Blood diseases (sickle cell, hemophilia)

Question 34

When is somatic cell CRISPR useful?

Answer 34

Any cell not included in reproduction.

Question 35

When is germline cell CRISPR useful?

Answer 35

Cells in reproductive line.