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Microbio & Biofilms > CRISPR-Cas > Flashcards

CRISPR-Cas Flashcards

1
Q

Clustered regularly interspaced short palindromic repeats (CRISPR)

A

Antivirus systems in bacteria
Prevent replication - cleave DNA and RNA
prevent spreading - dormancy and suicide

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2
Q

Host virus arms race with CRISPR

A

Infection - phage wins
No infection - bacterium wins due to host CRISPR-Cas system
Infection phage wins - virus anti CRISPR system
No infection bacterium wins - host anti anti CRISPR system

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3
Q

CRISPR-Cas mechanism

A

Foreign DNA acquisition in repeat region (AT rich) In spacer

Cas genes complete CRISPR locus
CRISPR RNA processing - pre CRISPR RNA (pin region where repeat is)

RNA guided targeting of viral element - loading of RNA and match virus the CAs9 cleaves

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4
Q

CRISPR array

A

Memory of adaptive immune system
Can be used to identify bacterial hosts - spacers can be matched in libraries that shows what bacteria have had the virus before

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5
Q

PAM (protospacer adjavent motif)

A

Allows for self/non self discrimination to avoid auto immunity (by targeting its own genome)

CRISPR don’t have auto immunity because of this as host won’t have PAM near motif
Helps make search quicker
NGG = PAM of

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6
Q

CRISPR class 1

A

Multi protein effective (cascade) complexes
Case 3, 8 or 10 (large subunit), 11 (small subunit), 7, 5, 6, 1, 2, 4, spacers and CRISPRs

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7
Q

CRISPR class 2

A

Single protein effector complexes
Cas 9,12 or 13 (single effector protein)c cas 1, 2, 4, spacers and CRISPRs

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8
Q

Class 1 CRISPR-Cas systems types

A

Type 1, 3 and 4

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9
Q

CRISPR Class1 type 1

A

Cas3-Cascade
Type 1 cascade

Binds and unwinds complementary dsDNA

Cas3- nuclease so cleaves DNA

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10
Q

CRISPR class 1 type 3

A

Cas10 -cascade
Type 3 Csm/Cmr

RNA guided, RNA cleaving system
Target activated ssDNA cleavage
Target activated cyclic oligoadenylated synthesis

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11
Q

CRISPR class 1 type 4

A

CSF-cascade

Target nucleic acid?
Subunit stoichiochemtry?

Not fully understood

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12
Q

CRISPR Class 2

A

Type 2, 5, 6

Genome engineering
Biotechnology (cas9, 12, 13)
Cas 1 and 2 proteins aiding on cleavage

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13
Q

CRISPR Class 2 type 2

A

CAs9
Binds and cleaves complementary dsDNA
Effector complex

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14
Q

CRISPR Class 2 type 5

A

Cas 12
Binds and cleaves complementary DsDNA

Type 5 (subtype V-A) effector complex

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15
Q

CRISPR Class 2 type 6

A

Cas 13
Non specific cleavage of complementary ssRNA
Collateral ssRNA degeneration (host and viral)

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16
Q

Class 1 and 2 CRISPR-Cas systems are distinguished based on?

A

Effector molecule: Class 1 systems have multi protein effector, class 2 have single protein effector

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17
Q

What does CRISPR-Cas immunity rely on?

A

Programmable Sequence specific nucleases
But nucleic acid cleavage is not limited to target site specified by crRNA

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18
Q

Cas13a (class 2 type 6)

A

2 HEPN nuclease domains
Binding to target RNA to crRNA causes conformational change that brings domains closer so active HEPN site
Active site cleaves RNA in sequence independent manner

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19
Q

CRISPR deference by collateral damage

A

Caused by cleavage if RBA in sequence independent manner

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20
Q

Cas 12a (class 2 type 5)

A

RuvC domain responsible for nucleolytic activity
Blocked by Rec lobe

Binding, conformational change, activates RuvC nuclease domain, cleavage of target DNA and non specific ssDNA

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21
Q

Class 1 type 3A collateral damage defence

A

HD and palm domains Inactivated to limit toxicity

HD domain (non specific cleavage of ssDNA)
Palm domain (atp to 4 or 6 coA)
cOA = secondary messenger activated Csm6 by binding CARF domain activating HEPN domain (non specific RNA cleavage)
Target recognition by type 3 effector complexes = cleavage of RNA complementary to crRNA so inactivate HD and palm domains so limit toxic effects of depredation

22
Q

Steps of defence by class 1 type 3A

A

Specific cleavage if phage RNA (by csm3)
Non specific cleavage of host RNA: dormancy (by csm6)
Non specific cleavage of host DNA: suicide (HD domain of cas 10)

23
Q

Strategies of CRISPR cas

A

Diverse defence system of bacteria and archaea
Strategy 1- protect infected cell by specific targeting phage DNA/RNA
Strategy 2- protect population by sacrificing infected cell (dormancy or suicide)

All systems are RNA GUIDED

24
Q

What systems target DNA

A

Cas 3 cascade (type 1 class 1)
CAs9 (type 2 class 2)
cas12 (type 5 class 2)

25
Q

What systems target RNA

A

Cas 10 cascade (type 3 class 1)
Cas 13 (type 6 class 2)

26
Q

How is collateral damage activated in type 3, 5, 6 CRISPR cas systems?

A

By degradation of target RNA or DNA

27
Q

CRISPR engineering examples

A

Gene drives
CRISPRcas tech
Epigenome engineering

28
Q

CRISPR cas engineering - cas 9

A

Cas 9 most widely used genome editing tool
In engineered CRISPR cas9 systems, cas9 interacts with backbone of guide RNA
Complementary pairing of spacer portion of gRNA to DNA next to Pam causes blunt DNA double strand break by domains RuvC and HNH

29
Q

CRISPR cas engineering - cas 12 a

A

Recognise DNA target sequence
Target recognition results in generation of staggered DNA double strand break by RuvC domain and Nuc domain

Good for integrating DNA sequences in precise orientation

30
Q

How do gene editing nucleases function?

A

Generate targeted DNA breaks that induce DNA damage response and stimulate repair by various endogenous mechanisms

Different DNA repair mechanism allow development of specific genome editing strategies

31
Q

Non homologous end joining (NHEJ) mediated repair

A

Repair of double stranded breaks (cas9 and 12)

Small insertion or deletion mutations
Large targeted deletions
Homologous independent targeted integrations

32
Q

Homologous directed repair (HDR)

A

Genome editing by providing either double or single stranded oligodeoxynucleotide (ssODN) donor templates that contain homologous arms to cut target site

Single nucleotide alterations
Insertion of larger sequences

33
Q

Single base editing

A

Most common genetic variants associated with disease are point mutations
For single nucleotide conversion (c to r or g to a) cas9 nickase fuses go cytidine deaminase eg APOBEC1

Increased base editing efficiency, 2 uracil glycosylase inhibitors (UGIs) fused to base editor for prevention of cellular base excision repair

34
Q

Cas3 cascade - precision DNA editing

A

Target recognition, cascade recruits cas3 to generate single stranded nick followed by 3 to 5 degredation if target DNA

CAS3 = antimicrobial tool by directing to bacterial genomes for degredation and cell death

35
Q

RNA manipulation in living cells

A

CAs9 repurposed to target RNA by providing matching gRNA and complimentary Pam

CAs9 ortholgues - target RNA in absence of PAM

Cas13 - target and non target RNA molecules

36
Q

Catalytically inactive applications

A

RNA visualisation and tracking

Adenosine deaminase acting on RNA fuses catalytically deficient cas for base editing to correct disease relevant mutations

37
Q

Targeted RNA degradation

A

Cas13 used to target RMA degradation in eukaryotic cells for applications like targeting viral RNA or toxic RNA

Cas9 for hepatitis c

38
Q

CRISPRi

A

Transcription repression
Catalytically deficient cas9 (dcas9) or dcas9 fused with effectors like transcription repression domain of KRAB box target promoters, 5’ untranslated regions or enhancers

39
Q

CRISPRa

A

Transcription activation
Achieve by fusion of dcas9 to transcription activation domains
Activated expression of specific genes

40
Q

Most sensitive and specific diagnostic tests

A

Detection of nucleus acids
Cas12a and cas13a collateral cleavage explored for diagnostics

41
Q

CRISPR diagnostics for pathogen detection

A

Pre amplification of DNA or RNA
RNA targeting CRISPR enzymes (cas13a), amplified proctor is t7 transcribed into RNA
Binding of crRNA to complementary target sequence activated cas enzyme
Triggers collateral cleavage of fluorescent reporters

Cas13a (used in Sherlock - RNA)
Cas12a ( used in DETECTR - DNA)

42
Q

What enzymes are used in CRISPR gene/genome editing?

A

Cas9
Cas12
Cas3 cascade

43
Q

functions of CRISPR-cas systems

A

Intertwined in repair processes
Immunity and cell death
Signal transduction pathways
Regulation of microbial gene expression and virulence

44
Q

Non canonical functions of CRISPR cas

A

Evasion of host immunity
Regulation of biofilm formation

45
Q

Evasion of host immunity using CRISPR cas

A

Cas9 + tracrRNA + scaRNA target endogenous transcript encoding immuno simulators bacterial lipoprotein (BLP) - mRNA degradation and decreased transcript levels

Increased BLP levels activate toll like receptor 2 dependent proinflammatory response. Attenuation of bacteria during infection

Critical to francisella novicida infection

46
Q

Regulation of biofilm formation using CRISPR cas

A

In pseudomonas aeruginosa, CRISPR cas modulates biofilm formation
Lysonogised, CRISPR Cas interacts with gene in prophage to inhibit formation of biofilms

Salmonella, cas3 targets and downregs expression of genes involved in degradation of quorum drinking molecules so increased biofilm formation

47
Q

Anti CRISPR proteins (Acr)

A

“Kamakazi attack”
Phage derived small protein inhibitors of CRISPRcas systems
Some are specific some not

48
Q

Mechanisms of Acr function

A

1) inhibitors of target DNA binding eg acrIIA4 (occluded cas9 Pam recognition domain)
AcrIIC3 (cas9 dimerisation)

2) inhibitors of DNA cleavage eg
AcrIIC1 (disables cas9 by binding to HNH domain)
AcrIE1 (blocks DNA cleavage by binding to cas3)

49
Q

Applications of Acr

A

Decrease cleavage of DNA or RNA off targets
Reduce cell cytotoxicity by restricting expression of cas9
Restricting gene drive
Controlling transcription
Gene silencing
Controlling gene imaging
Detection affirm fir cas complexes
Bacteriophage therapy

50
Q

A Pam is?

A

3-5 bp sequence adjacent to phage in CRISPR array

51
Q

What is the role of crRNA in CRISPR cas?

A

Ensure cas enzyme cuts at right point in phage genome

52
Q

Which CRISPR cas system induce collateral damage upon target recognition

A

Type 3 (cas10 cascade)
Type 5 (cas12)
Type 6 (Cas13)

Microbio & Biofilms (8 decks)

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