Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Regulation Of Gene Expression > Lesson 10 > Flashcards

Lesson 10 Flashcards

1
Q

what is gene addition?

A

provide patients cells with a normal copy of a gene - aberrant gene still remains

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what is the goal of genome editing?

A

to correct a gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is another term for substitution in normal physiology?

A

homologous recombination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

naturally, how does homologous recombination occur?

A

at very low frequencies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

in regards to zinc finger and other meganucleases, what does a nuclease refer to?

A

a molecule that is able to cut DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what are meganucleases?

A

different versions derived from zinc finger

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what does TALEN stand for?

A

transcription activator like effector nuclease (cuts DNA and the genome)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what does CRISPR stand for?

A

clustered regularly interspaced palindrome repeat

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

if the sequence is known and you have information on the transcriptome and you want to target DNA, what systems could you use?

A

TALEN and Cas9

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

if you have the genome and gene information with the annotated transcription start site, what systems can you use?

A

all technologies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what is one consideration when it comes to genome editing that is very important but not scientific?

A

the cost

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what is something you must be very careful of when using editing molecular tools?

A

the systems are very efficient → you have the probability of perturbing the genome or changing the sequence of DNA also in other portions of the genome (off target)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what is the very fist step in any genome editing process?

A

cutting the double strand in DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what does the editing by nuclease induce?

A

a double stranded break (DSB)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what is non-homologous end joining (NHEJ)?

A

the cut ends are repaired by the cellular machinery

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what is a risk of NHEJ?

A

the re-joining of ends is not precise - there is a possibility to have some deletions or insertions of bases (indels)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

in most cases, what does NHEJ cause?

A

a disruption in the function of the gene (change coding frame) creating a knock out gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what is an inversion, and what occurs?

A

if your nuclease cuts twice in the genome in close positions, there is flipping of the piece of the genome and it is reinserted

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what is HDR?

A

homology direct repair → adds a piece of DNA in a very precise location in the genome

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

what must be provided when doing HDR?

A

a donor template

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

what is another name for the substitution that occurs in HDR?

A

targeted gene addition → you know exactly where to put your gene and is gene correction through substitution

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

what is the advantage to HDR?

A

if you do gene addition of just the coding region targeting the promoter of the gene (that naturally expressed that gene) → you will have the expression in a more physiological way from the endogenous promoter and not by adding exogenous promoter to your gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

between NHEJ and HDR, what is the preferred method?

A

NHEJ → occurs naturally at high frequencies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

what is the disadvantage to HDR?

A

in order to preform gene substitution you need to perform HDR at a very high frequency → occurs at a very low frequency naturally, even with the addition of nucleases it is less efficient compared to NHEJ

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

what is the zinc finger motif?

A

a DNA binding domain

26
Q

what is the nuclease domain in ZFN provided by?

A

a restriction enzyme called Fok1

27
Q

why are ZFN considered to be modular DNA recognition proteins?

A

the nucleases do not occur naturally in the body, and therefore can be designed

28
Q

what are ZFN efficient in?

A

inducing genome sequencing changes in almost all types of cells

29
Q

what is a con of ZF?

A

there are some difficulties in designing how the protein will be and in validating how the ZFNs is very effective or not in cutting your target

30
Q

what is an important rule to remember about zinc finger?

A

ZFN work in couples → you always have to design 2 ZFNs depending on your target site

31
Q

how do both ZFN bind to the target site?

A

as dimers (right and left ZFN)

32
Q

where to TALEN proteins come from?

A

they are not present in nature, but derived from a natural molecule which is the transcriptional activator-like effectors (TALE)

33
Q

where do TALE proteins naturally occur?

A

present in bacteria that infect plants

34
Q

what is the function of TALE in plants?

A

they are activators of transcription that are injected into the plant cell to activate the expression of genes that help the bacteria for replication

35
Q

how do we go from TALE to TALEN?

A

TALE is a fusion protein between TALE without the terminal translocation domain, this is deleted, we do have the nuclear localization domain (because we need to target DNA), we have the deletion of activator domain (we don’t need to activate gene) and replacement of this part with the nuclease.

36
Q

what does TALEN work as?

A

a dimer (like ZFN)

37
Q

what does TALEN offer compared to ZFN?

A

offers the possibility to have a longer recognition sequence for DNA

38
Q

what does having a longer recognition sequence for DNA mean?

A

you can more precisely target a gene because you increase the length of the recognition domain

39
Q

what is a major disadvantage to TALEN?

A

they are very difficult to synthesize and the design/synthesis is not very efficient

40
Q

between ZFN and TALEN, which is usually chosen?

A

ZFN

41
Q

when we use the HDR pathways for ZFN, what diseases can we treat?

A

X-linked SCID (immunodeficiency) (theoretically for Haemophilia B Sickle cell disease but has not yet worked)

42
Q

give an example of a case when we want to disrupt the gene and not correct it → ZFN with KO via NHEJ?

A

HIV resistance by CCR5 knockout in T cells (clinical trial) → if we target one of these molecules of the receptor which favor the entry of HIV in cells (CCR5) and disrupt and make a knock-out of CCR5, in this case we’ll provide the cells a tool to resist HIV infection.

43
Q

what is another clinical trial being used with the NHEJ pathway and ZFN?

A

KO of endogenous TCR genes in the generation of tumor-specific T-cells, used in oncology

44
Q

describe the bacterial vaccination system:

A

many bacteria use small noncoding RNAs to protect themselves from viruses → in this way bacteria cells have memory of infections because they carry a record of past exposure that is used to protect against future exposures

45
Q

what is the first step that occurs when a virus is injected into a bacterium?

A

the short viral DNA sequence is integrated into the CRISPR locus

46
Q

after the viral DNA is integrated into the CRISPR locus, what occurs?

A

the RNA is transcribed from the CRISPR locus and the pre-crRNA is generated, transcribed, and then bound to Car proteins

47
Q

when a new viral infection occurs, what happens?

A

the small crRNA in the complex with Cas seeks out and destroys viral sequences

48
Q

what is the CRISPR-Cas9 system based on?

A

antisense RNAs as memory signatures of past invasions

49
Q

what is the CRISPR-Cas9 RNA-endonuclease complex based on?

A

the adaptive immune system of Streptococcus Pyogenase SF370

50
Q

how does the CRISPR system target and cleave its target DNA?

A

though a programmable short guide RNA (which we can customize) binding to its complementary DNA strand and a Cas9 protein

51
Q

WRITE OUT MOLECULAR PROCESS OF CRISPR-CAS9

A
52
Q

when engineering the CRISPR-Cas system, what do we keep from the natural system to begin the process?

A

keep only two portions, the crRNA and the trascrRNA, and we make a fusion of them, obtaining the sgRNA, which is the single-guide RNA

53
Q

the single-guide RNA (sgRNA) can form a complex with Cas9, targeting the DNA site for cleavage → where does Cas9 cleave the DNA?

A

at the PAM sequence (protospacer adjacent motif)

54
Q

when designing a guide RNA, what must be present in the target DNA?

A

a PAM sequence in order to be recognized by the Cas protein

55
Q

what does Cas9 target?

A

targets genomic sequences containing the tri-nucleotide (NGG, which N can correspond to A, T, C or G) protospacer adjacent motif (PAM) and complementary to the gRNA (guide-RNA)

56
Q

what can Cas9 be programmed to recognize?

A

virtually any genes though the manipulation of the gRNA sequence

57
Q

what is the NGG required for?

A

to allow the cutting of the DNA (very frequent in the genome)

58
Q

what is the role of NGG PAM?

A

following binding to target site, Cas9 cleaves DNA about 3 or 4 bp upstream of the NGG PAM

59
Q

after NGG PAM cleaves DNA, how are the double strands generated repaired?

A

the double strands generated are repaired by either NHEJ or HDR

60
Q

what is required in order to reprogram the CRISPR-Cas9 system for new targets?

A

the presence of PAM in the target DNA and changing the first ~20 bases in the guide RNA

61
Q

when a donor template is given, what can CRISPR-Cas9 achieve?

A

almost precise genome correction

Regulation Of Gene Expression (28 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions