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Biofilms & Microbial communities > CRISPR-Cas > Flashcards

CRISPR-Cas Flashcards

Origins and evolution

1
Q

what are the two outcomes of CRISPR-Cas activity

A

prevent replication - cleave DNA and RNA
prevent spreading - dormancy and suicide

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2
Q

what is the host-virus arms race?

A
  • The ability of the bacteriophage to continue creating pressure over the cell, the cell creating new ways to overcome the responses
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3
Q

what is the CRISPR array?

A

the memory of the adaptive immune system
Consist of two to several hundred direct repeats (typically 25–36 bp in size) separated by spacers, some of which are homologous to segments of virus or plasmid genomes.

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4
Q

what is the CRISPR locus?

A

A typical CRISPR-cas locus consists of a CRISPR array and adjacent cas genes that form one or more operons

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5
Q

what are the three steps of CRISPR-Cas immunity

A
  1. foreign DNA acquisition
  2. CRISPR RNA processing
  3. RNA-guided targeting of viral element
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6
Q

what is the PAM?

A

PAM = protospacer adjacent motif, allows for self/non-self discrimination to avoid auto-immunity

in the viral DNA!

a motif close to bound target but not part of the cleavage material
follows the protospacer

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7
Q

what is the major difference between class 1 and class 2 CRISPR-Cas systems?

A

class 1 uses multi protein effector cascade complexes and class 2 uses single protein effector complexes

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8
Q

give three class 1 CRISPR systems

A

Types 1, 3 and 4

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9
Q

what makes up the interference module of class 1 CRISPR systems

A

the effector module (crRNA and target binding)
and target cleavage

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10
Q

what is crRNA?

A

the transcript of the CRISPR locus
it is processed by Cas proteins - mature crRNA transcripts contain a partial conserved section of repeat and a sequence spacer that is complementary to the target DNA

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11
Q

what is tracrRNA

A

trans-activating CRISPR RNA
binds crRNA by complementary base pairing in type 2 CRISPR systems to guide Cas9

Type II CRISPR-Cas systems require a tracrRNA which plays a role in the maturation of crRNA.[3] The tracrRNA is partially complementary to and base pairs with a pre-crRNA forming an RNA duplex. This is cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid

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12
Q

breifly describe how the type 1 CRISPR system works

A

The crRNA can form a complex with the Cas proteins in the Cascade and scan the genome until it finds the PAM, then unwinds the DNA forming an R loop, guiding the complex to the target DNA sequence.
Cas3 is recruited for the nuclease-helicase activity.

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13
Q

what cleaves the DNA in a type 1 CRISPR system?

A

Cas3

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14
Q

what are the roles of Cas5, Cas6 and Cas7 in type 1 CRISPR systems?

in the Cascade

A

Typically in the Cascade, Cas6 generates the mature crRNAs while Cas5 and Cas7 process and stabilize the crRNA

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15
Q

what is Cascade?

A

CRISPR-associated complex for antiviral defense

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16
Q

what is the type 1 CRISPR system characterised by

A

Type-I CRISPR systems are characterized by Cas3, a nuclease-helicase protein, and the multi-subunit Cascade

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17
Q

what type of nucleic acids do type 1 CRISPR systems target?

A

dsDNA

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18
Q

what are type 3 CRISPR systems characterised by?

A

Type-III CRISPR systems are characterized by Cas10, an RNA cleaving protein
and csm/cmr complex

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19
Q

what kind of nucleic acids do type 3 CRISPR systems target?

A

RNA
(but has capacity to target ssDNA too)

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20
Q

once bound to target sequence, what activity is initated by the complex in type 3 CRISPR systems

A
  • RNA cleavage
  • ssDNA cleavage
  • cyclic oligoadenylate synthesis

creatign death and dormancy

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21
Q

what crispr system involved Csm/Cmr complex

A

type 3

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22
Q

what crispr system requires tracrRNA

A

type 2

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23
Q

every protein that has nucleic acid binding capacity has what domain

A

helix turn helix domain

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24
Q

breifly describe how type 2 CRISPR systems work

A

crRNA and tracrRNA form a complex known as the guide RNA (gRNA)
The crRNA within the gRNA is what matches up with the target sequence or protospacer after the PAM is found. Once the match is made Cas9 will make a double-stranded break.

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25
Q

what is the PAM sequences for type 2 CRISPR systems?

A

NGG

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26
Q

what domain guide the dsDNA break in type 2 CRISPR systems

A

RuvC

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27
Q

what type of nucleic acids do type 2 crispr systems target

A

dsDNA

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28
Q

what two CRISPR systems use RNA guided RNA cleavage

A

type 3 and 6

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29
Q

what are type 2 crispr systems characterised by

A

Type-II CRISPR systems are characterized by the single signature nuclease Cas9.

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30
Q

what nucleic acids can cas12 cleave

A

ssDNA, dsDNA, and RNA

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31
Q

what is the effector nuclease of the type 5 CRISPR systems

A

Cas12

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32
Q

how does collateral damage occur in type 6 CRISPR systems

A

via non specific cleavage (ssRNA) following recognition of target sequence

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33
Q

what is the function of the HEPN domains in type 6 CRISPR systems?

A

nucleic acid cleavage

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34
Q

describe how the effector cleaves the RNA in type 6 CRISPR systems

A

Cas13a possesses two HEPN nuclease domains
Binding of target RNA to the crRNA induces a conformational shift that brings the domains in close proximity to form an active HEPN site.
The active site then cleaves RNA in a sequence-independent manner.

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35
Q

what domain is responsible for the nucleolytic activity in Cas12a

A

RuvC

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36
Q

describe how the effector cleaves the DNA in type 5 CRISPR systems

A

Upon binding of ssDNA or dsDNA targets to the crRNA , the effector undergoes a conformational shift that exposes and activates the RuvC nuclease domain to license cleavage of the target DNA and non-specific cleavage of ssDNA

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37
Q

what obscures the RuvC domain of Cas12a before target recognition?

A

the Rec lobe

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38
Q

what are the subclasses of type 3 CRISPR systems?

how are they distinguished

A

type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets respectively

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39
Q

target recognition activates what two domains of Cas 10

A
  • HD domain
  • Palm domain
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40
Q

whats the purpose of the HD domain in Cas 10

A

non-specifically cleaves ssDNA

The ssDNA degraded is confined to the vicinity of the target sequence, because it is part of the transcription bubble formed after RNA polymerase synthesizes the target RNA. Proximity of the complex degrades nonspecifically the ssRNA.

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41
Q

what is the purpose of the palm domain in Cas10

A

converts ATP into 4 or 6 member rings of cyclic oligoadenylates (cOA). cOA acts as a secondary messenger and activates Csm6

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42
Q

how does cOA cause collateral nucleic acid degredation in type 3a CRISPR systems?

A

cOA acts as a secondary messenger and activates Csm6 by binding to its CARF domain causing activation of the HEPN domain unleashing non-specific RNA cleavage.

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43
Q

how is the toxicity of collateral nucleic acid degradation limited in type 3a CRISPR systems

A

Target recognition by the type III effector complexes triggers the cleavage of the RNA complementary to the crRNA by the Csm3 subunits of the complex, causing the inactivation of both the HD and Palm domains of Cas10, an event that limits the possible toxic effects of the collateral nucleic acid degradation carried on by type III systems

44
Q

non specific cleavage of host DNA leads to …

A

suicide

45
Q

non-specific cleavage of host RNA leads to …

A

dormancy

46
Q

what performs specific cleavage of phage RNA in type 3a CRISPR systems?

A

csm3

47
Q

what are the two strategies taken by CRISPR-cas to protect the host

A
  • strategy-1 is to protect an infected cell by specific targeting phage DNA or RNA
  • strategy-2 is to protect a population by sacrificing the infected cell(s) (dormancy or suicide)
48
Q

All CRISPR-Cas systems use CRISPR-derived … guides to identify the target to cleave

A

crRNA

49
Q

what CRISPR systems target DNA

3

A

Cas3-Cascade (Type I, Class-1)
Cas9 and Cas12 (Type II and Type V, Class-2)

50
Q

what CRISPR systems target RNA?

2

A

Cas10-Cascade (Type III, Class-1)
Cas13 (Type VI, Class-2) use guide to target RNA

51
Q

what CRISPR system performs non specific cleavage of host RNA and DNA

A

Type III (Cas10-Cascade)

52
Q

what CRISPR system performs non specific cleavage of host DNA only

A

Type V (Cas12)

53
Q

what CRISPR system performs non specific cleavage of host RNA only

A

Type VI (Cas13)

54
Q

what industries can CRISPR engineering be utilised in?

A
  • gene surgery
  • drug development
  • animal models
  • epigenetic variation
  • materials
  • food
  • fuel
55
Q

how does Cas9 work in engineered systems?

A

In engineered CRISPR–Cas9 systems, Cas9 interacts with the backbone of the guide RNA (gRNA).
Complementary pairing of the spacer portion of the gRNA to a DNA target sequence positioned next to a PAM results in generation of a blunt DNA double-strand break by the two Cas9 nuclease domains, RuvC and HNH.

56
Q

how does Cas12 work in engineered systems

A

Cas12 nucleases recognize DNA target sequences with complementarity to the CRISPR RNA (crRNA) spacer positioned next to a 3ʹ PAM.
Target recognition results in the generation of a staggered DNA double-strand break by a RuvC domain and a putative nuclease (Nuc) domain
This allow engineering in precise orientation

57
Q

why is the production of staggered ends more advantageous for engineering applications?

A

helps in integrating DNA sequences in a precise orientation

58
Q

beyond the activity of the nucleases, what is important for the gene editing functions of CRISPR

A

These gene editing nucleases function by generating targeted DNA breaks that induce the DNA damage response and stimulate repair by various endogenous mechanisms.

59
Q

what ways does non homologous end joinging (NHEJ) mediated repair help in genetic engineering?

3

A

while repairing the DNA…
1. Induces small insertion or deletion mutations (indels)
2. Large, targeted deletions can be produced through repair between two double-strand breaks produced by simultaneous targeting of nucleases to two genomic sites
3. homology-independent targeted integrations can be directed to a single cut site by providing donor DNA that is independently targeted for cutting

60
Q

what is non homologous end joining?

A

a pathway that repairs double-strand breaks in DNA
the break ends are directly ligated without the need for a homologous template,

61
Q

what is needed for homology directed repair in genome editing?

A

genome editing by providing either double-stranded or single-stranded oligodeoxynucleotide (ssODN) donor templates that contain homology arms (grey rectangles) to the cut target site.

62
Q

what gene editing purposes can homology directed repair be utilised for?

A

Single-nucleotide alterations
Insertion of larger sequences

63
Q

what are the most common genetic variants associated with human disease

A

point mutations

64
Q

how can CRISPR based genetic engineering be used to create single nucleotide (C>T/ G>A) conversions

A

Cas9 nickase has been fused to cytidine deaminases such as APOBEC1.

thsi causes cytosine deamination and nicking, followed by cellular mismatch repair then DNA replication/repair that results in a targeted nucleotide alteration

For increased base-editing efficiency, two uracil glycosylase inhibitors (UGIs) have been fused to a base editor for prevention of cellular base excision repair.

65
Q

how is base editing efficiency increase in single base editing by Cas9/cytosine deaminase fusion protein

A

For increased base-editing efficiency, two uracil glycosylase inhibitors (UGIs) have been fused to a base editor for prevention of cellular base excision repair.

66
Q

hwo can Cas3-Cascade be used as an antimicrobial tool (gene editing)

A

Following target recognition, Cascade recruits Cas3 to generate a single-strand nick, which is followed by 3ʹ to 5ʹ degradation of the targeted DNA.
The unique cutting mechanism of Cas3 is being harnessed as an antimicrobial tool by directing type I systems to bacterial genomes for degradation and subsequent cell death.

67
Q

how can Cas9 be repurposed as an RNA targetting tool?

A

Cas9 was repurposed to target RNA (RCas9) by providing it with a matching gRNA and a complementary PAM-presenting oligonucleotide (PAMmer) to create a double stranded nucleic acid that can be cut by Cas9

68
Q

why is a PAMmer not necessary for Cas9 orthologues to target RNA

A

Cas9 orthologues exist that can naturally target RNA in the absence of a PAMmer

, thereby demonstrating PAM-independent RNA cleavage

69
Q

how can Cas13 be used to demonstrate the function of proteins in cells

A

Catalytically deficient Cas13 maintains the capacity to bind to the targeted RNA, and therefore blocks the function

70
Q

how can CRISPR be used for RNA visualisation and tracking

A

a fluorescent protein can be fused to the catalytically deficient Cas and co-localize with an array of crRNAs or gRNAs

71
Q

how can catalystically inactive Cas be used to correct disease relevant mutations

A

Adenosine deaminase acting on RNA (ADAR) can be fused to catalytically deficient Cas for RNA A→I base editing to correct disease-relevant mutations.

72
Q

give three catalytically inactive applications

A
  • block protein targets to reveal function
  • RNA visualisation and tracking
  • base editing to correct disease relevant mutations
73
Q

what applications are there for using Cas13 for targeted RNA degradation

A

targeting viral RNA or toxic RNAs that contain microsatellite repeat expansions

74
Q

what has been used to target the RNA genome of hepatitis C virus?

A

Francisella novicida Cas9 has been repurposed in eukaryotic cells to target the RNA genome of hepatitis C virus

75
Q

what is CRISPRi

A

CRISPR interference (CRISPRi) is a genetic pertubation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells.

76
Q

how is transcription repression performed by CRISPRi?

A

Catalytically deficient (dead) Cas9 (dCas9) alone or dCas9 fused to effectors such as the transcription repression domain of Krüppel-associated box (KRAB) can be targeted to promoters, 5ʹ untranslated regions (UTRs) or enhancers.

77
Q

what is CRISPRa?

A

CRISPR activation
the ability to increase expression of genes in their native context
uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs.

78
Q
A
79
Q

how does CRISPRa work?

A

Can be achieved by fusion of dCas9 to transcription activation domains to activate the expression of specific genes.
Add domains that cause binding to promoter domains

80
Q

how can CRISPR be used for diagnostics?

A

Pre-amplification of DNA or RNA
For RNA-targeting CRISPR enzymes (Cas13a), the amplified product is T7-transcribed into RNA
Binding of the crRNA to the complementary target sequence activates the Cas enzyme and,
triggers collateral cleavage of quenched fluorescent reporters.
fluorescence upon cleavage indicating target is present

Collateral damage only takes places upon successful cleavage of specific recognition site.
Fluorescence only upon cleavage

81
Q

what diagnostics system uses Cas13a?

A

SHERLOCK

82
Q

what diagnostics system uses Cas12a?

A

DETECTR

83
Q

what are the two fundamental ideas that enable CRISPR diagnostics?

A
  • Collateral damage only takes places upon successful cleavage of specific recognition site.
  • Fluorescence of reporter DNA/RNA only upon cleavage
84
Q

which cas proteins have been repurposed for diagnostics?

A

Cas 12 and 13

85
Q

in addition to adaptive immunity, what other functional networks are CRISPR-Cas systems involved in?

A
  • CRISPR–Cas activity is intertwined with repair processes.
  • CRISPR–Cas immunity appears to be coupled to programmed cell death.
  • CRISPR–Cas systems are connected to signal transduction pathways.
  • CRISPR–Cas systems contribute to regulation of microbial gene expression and virulence.
86
Q

give an example of a bacterium that uses CRISPR fo evasion of host (human) immunity

A

Francisella novicida

87
Q

describe how CRISPR is used to evade host immunity

A

Cas9 + tracrRNA + a small RNA termed scaRNA, target an endogenous transcript encoding an immuno-stimulatory bacterial lipoprotein (BLP).
In the absence of this regulation, increased BLP levels trigger the activation of a Toll-like Receptor 2 (TLR2)-dependent proinflammatory response, and result in complete attenuation of the bacteria during infection.

88
Q

how does CRISPR-Cas modulate biofilm formation in Pseudomonas aeruginosa?

A

When P. aeruginosa is lysogenized by a specific phage, the CRISPR-Cas system interacts with a gene in the chromosomally integrated prophage to inhibit the creation of biofilms.

89
Q

how does CRISPR-Cas regulate biofilm formation in Salmonella?

A

Cas3 targets and downregulates the expression of genes involved in the degradation of quorum sensing molecules, which results in promotion of biofilm-forming-related genes, leading to biofilm formation.

90
Q

what happens when you delete the Cas3 gene in salmonella?

A

Deletion of the cas3 gene made Salmonella less pathogenic to host cells and chickens.

91
Q

how can you target the CRISPR cas system to decrease the virulence of salmonella

A

Regulatory compounds could be designed and synthesized to block the expression of the cas3 gene to decrease the virulence of bacteria.

92
Q

what can be used to avoid off-target effects of CRISPR-Cas?

A

Anti-CRISPR (Acr) proteins

93
Q

what are anti-crispr (Acr) proteins?

A

Acr proteins are phage-derived small protein inhibitors of CRISPR-Cas systems that help phages evade the CRISPR-Cas immune system of bacteria.

94
Q

what is the significance of aca genes in acr transcription

A

an acr associated promoter drives high levels of acr transcription immediately after phage DNA injection, and Aca proteins subsequently repress this transcription. Without Aca activity, this strong transcription is lethal to a phage.

95
Q

what are the two main mechanism of anti-crispr (acr) proteins?

A
  • inhibition of target DNA binding
  • inhibition of DNA cleavage
96
Q

what applications are there for Acrs

A
  • reducing off targets
  • reducing cell cytotoxicity
  • restricting gene drive
  • controlling transcription
  • gene silencing
  • controlled gene imaging
  • detection platform for Cas complexes
  • bacteriophage therapy
97
Q

how can Acrs be used to reduce cell cytotoxicity?

A

Reducing cell cytotoxicity by restricting the expression of Cas9.
To prevent self-cleavage during the production of the viral vector, Cas9 activity is inhibited by AcrIIA2 and AcrIIA4 expressed from the producer cell and helper virus.

98
Q

how can Acrs be used to restrict gene drive?

A

Gene drive uses Cas9 to cut a chromosome to induce the cell to repair the break and in doing so to copy the drive gene to the damaged chromosome and spread it through the population

99
Q

what is gene drive?

A

a genetic element that introduces a bias in the relative chance of inheritance between distinct versions of a set of genes, enabling one to spread rapidly in a population at the expense of others even if it is disadvantageous to the organism.

100
Q

how can Acrs be used to control transcription?

A

Acrs that block target DNA binding can prevent the targeted function of the fusion protein at the desired genes or genes

101
Q

How can Acrs be used for gene silencing?

A

Binding of Acrs can turn Cas9 catalytically inactive without impairing its DNA-binding ability. The Cas9–Acr complex blocks the binding of RNA polymerase, thus interfering with transcription.

102
Q

how can Acrs be used for controlling gene imaging?

A

Fusion of an Acr with a photosensor protein such as LOV2, which is degraded by light, restricts dCas9 function to the presence of light

103
Q

how can Acrs be used for bacteriophage therapy?

A

In bacteria, Acrs inactivate CRISPR–Cas systems that include spacers targeting phages to increase the efficacy of phage therapy against bacteria

104
Q
A
105
Q
A

Biofilms & Microbial communities (11 decks)

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