L7 Gene Editing II

Question 1

What must a gene-editing nuclease be able to do?

Answer 1

Be gene-specific or reprogrammable

Question 2

What system makes gene editing accessible to most labs?

Answer 2

CRISPR

Question 3

What does gRNA stand for?

Answer 3

guide RNA

Question 4

What is the role of gRNA in CRISPR?

Answer 4

it guides the nuclease to the target DNA sequence

Question 5

What is the natural origin of CRISPR?

Answer 5

A bacterial immune system

Question 6

How does CRISPR acquire specificity?

Answer 6

Through memory of prior infections

Question 7

When was CRISPR discovered?

Answer 7

In the 1980s

Question 8

What is the effector nuclease used in CRISPR?

Answer 8

Cas9

Question 9

What determines Cas9’s target specificity?

Answer 9

the guide RNA sequence

Question 10

What are two broad CRISPR system classes?

Answer 10

class 1 and class 2

Question 11

what characterises class 1 CRISPR systems?

Answer 11

multi-protein complexes

Question 12

what characterises class 2 CRISPR systems?

Answer 12

single-protein effectors

Question 13

What type of break does Cas9 induce?

Answer 13

double stranded DNA break

Question 14

What are crRNA and tracrRNA?

Answer 14

components of CRISPR RNA involved in targeting

Question 15

what does the spacer sequence in CRISPR RNA do?

Answer 15

it guides the system to the protospacer (target) sequence

Question 16

What part of CRISPR RNA is constant?

Answer 16

The repeat region

Question 17

What part of CRISPR RNA is variable?

Answer 17

The spacer region

Question 18

What is the single-guide RNA (sgRNA)?

Answer 18

a fusion of crRNA and tracrRNA into one molecule

Question 19

What protein binds to the sgRNA?

Answer 19

Cas9

Question 20

What sequence does Cas9 search for before binding?

Answer 20

PAM sequence

Question 21

What is the PAM sequence required by SpCas9?

Answer 21

NGG

Question 22

What does PAM stand for?

Answer 22

Protospacer adjacent motif

Question 23

What happens when Cas9 finds a PAM sequence?

Answer 23

it begins unwinding the DNA to check for complementarity

Question 24

Does Cas9 function as a helicase?

Answer 24

no

Question 25

What is formed when RNA binds to the DNA target?

Answer 25

an R-loop

Question 26

What sequence must match for cleavage to proceed?

Answer 26

The seed sequence

Question 27

What triggers Cas9 cleavage activity?

Answer 27

formation of a 20bp R-loop

Question 28

What are the two nuclease domains in Cas9?

Answer 28

RuvC and HNH

Question 29

What does the RuvC domain cut?

Answer 29

The non-target DNA strand

Question 30

What does the HNH domain cut?

Answer 30

The DNA strand complementary to the guide RNA

Question 31

What does Cas9 cleavage produce?

Answer 31

A double strand break

Question 32

What is dCas9?

Answer 32

A catalytically inactive form of Cas9

Question 33

What can dCas9 be used for?

Answer 33

gene regulation and epigenetic studies

Question 34

What CRISPR types are used in gene editing?

Answer 34

Types II and V

Question 35

What does Cas13 target?

Answer 35

RNA

Question 36

What does Cas14 modulate?

Answer 36

Gene expression

Question 37

Why are there different CRISPR subtypes?

Answer 37

They recognise different PAM sequences

Question 38

How is CRISPR delivered into eukaryotic cells?

Answer 38

Via viral vectors or transfection

Question 39

What is a feature of Cas12a (Cpf1)?

Answer 39

It recognised a 5'TTTN PAM

Question 40

What kind of cut does Cas12a make?

Answer 40

a staggered cut with a 5-nucleotide overhang

Question 41

How does Cas12a simplify targeting?

Answer 41

It produces and processes its own guide RNAs

Question 42

What can a single Cas12a transcript do?

Answer 42

Target multiple sites by producing multiple crRNAs

Question 43

Can Cas proteins search both DNA strands?

Answer 43

yes

Question 44

What limits where Cas9 can cut?

Answer 44

The presence of a suitable PAM sequence

Question 45

What base pair rule is followed in CRISPR targeting?

Answer 45

Guide RNA base pairs with the complementary DNA strand

Question 46

What assay detects successful CRISPR editing?

Answer 46

Mismatch cleavage assay

Question 47

What is a key step after CRISPR editing?

Answer 47

Clonal expansion to isolate edited cells

Question 48

How is HR used in CRISPR editing?

Answer 48

a repair template with homology flanks the break

Question 49

What method introduces CRISPR components into cells?

Answer 49

Transfection

Question 50

What complex is formed for editing using CRISPR-Cas9?

Answer 50

Cas9 bound to guide RNA

Question 51

What is the protospacer?

Answer 51

The DNA sequence targeted by the guide RNA

Question 52

What structure in RNA allows folding in CRISPR?

Answer 52

Hairpin loops

Question 53

Why is tracrRNA needed in natural CRISPR systems?

Answer 53

to bind the repeat sequence and process pre-crRNA

Question 54

What makes CRISPR versatile for labs?

Answer 54

The RNA can be easily reprogrammed to target any sequence

Question 55

What determines CRISPR-Cas9's editing location

Answer 55

PAM presence and guide RNA sequence

Question 56

Why might Cas9 not cut a target stite?

Answer 56

if there's no adjacent PAM sequence

Question 57

What is the benefit of using Cas12 over Cas9?

Answer 57

Smaller guide RNAs and staggered cuts

Question 58

What determines the CRISPR system type classification?

Answer 58

the number and type of effector proteins

Question 59

What structure does Cas9 form with gRNA?

Answer 59

a ribonucleoprotein complex

Question 60

What is the result of cleavage in both DNA strands by Cas9?

Answer 60

A blunt end double-strand break