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325 - World of RNA > 5 - Mechanisms of CRISPR Cas - Bose > Flashcards

5 - Mechanisms of CRISPR Cas - Bose Flashcards

1
Q

what does CRISPR stand for?

A

clustered regularly interspaced short palindromic repeats

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2
Q

what is the type and class of CRISPR system used in molecular biology? briefly describe this system

A

Class 2, type II CRISPR Cas9

- bacterial immune system that has been modified for genome engineering

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3
Q

CRISPR Cas is highly dependent upon the ____ and ____ of non coding RNA transcripts to create an ___ complex

A

expression
processing
RNP

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4
Q

why is CRISPR such a useful tool in molecular biology?

A

highly adaptable, countless variations of system for different uses

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5
Q

what are the 2 components of the CRISPR system? briefly describe them

A
  • consists of a guide RNA (gRNA) and non-specific CRISPR-associated nuclease (Cas9)
    gRNA;
  • short synthetic RNA composed of scaffold sequence necessary for Cas9 binding and a spacer sequence (around 20nt) which binds to the Target DNA
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6
Q

what is the prerequisite for the spacer target sequence?

A

immediately upstream of a PAM site

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7
Q

draw a simple diagram of the CRISPR Cas components and recognition of DNA

A

325 -5 word

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8
Q

what is the PAM site?

A
  • protospacer adjacent motif
  • present on the non target strand
  • PAM specific to the species of Cas present
  • eg spCas9 has PAM sequence NGG
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9
Q

where does the DSB occur on the DNA?

A

3nt upstream of the PAM site

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10
Q

describe the architecture of Cas9
- include the names of the lobes, where the spacer and scaffold sequences bind etc
DAG

A

RECOGNITION LOBE (REC);
- responsible for binding to scaffold of sgRNA
NUCLEASE (NUC);
- contains HINH (cleavage of TS) and the RuvC (cleavage of NTS) responsible for the DSB

Cas9 and gRNA form RNP complex;

  • gRNA scaffold binds in +vely charged groove
  • gRNA spacer remains accessible to bind target strand DNA
  • gRNA binding to Cas9 causes conformational changes which cause it to change from inactive conformation to active conformation (DNA binding)
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11
Q

describe how Cas9 specificity to the target arises?

A
  • Cas9 binding to the PAM site

- complementarity and base pairing between the gRNA spacer and the target strand

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12
Q

what are the ways of repairing this Cas9-induced DSBs? how is this useful?

A

NHEJ;
- error prone repair
- introduction of indels (insertion or deletion) causing frameshift or knockout mutations
HDR;
- exogenous DNA donor template supplied
- used for gene correction or knock in experiments
- include tagging of target proteins

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13
Q

how are Class 2 CRISPR systems characterised? name one type of this and name the Cas protein invovled

A
  • characterised by RNA guided effector complexes that only require a single mutlidomain subunit protein for interference (the Cas endonuclease)
  • eg Type II system, characterised by the cas9 gene
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14
Q

what is CRISPR Cas immunity?

A
  • bacteria and archae, Crispr Cas immunity provides defence against mobile genetic elements (MGEs) from phage infection
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15
Q

how has CRISPR evovled in bacteria?

A

ongoing competition between phages/MGEs and bacteria have created rapid evolution and diversification of the CRISPR loci

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16
Q

what does the CRISPR system look like in the genome? what are the components and what do they code for

A
  • CRISPR array (encoding noncoding RNA)

- neighbouring Cas genes (encoding protein)

17
Q

draw a diagram showing the OVERALL scheme of the CRISPR system in bacteria. In the diagram include an infecting bacteriophage

A

325 - 5 word

18
Q

name and describe the 3 steps in the CRISPR pathway and defence against MGEs

A

ADAPTATION;
- protospacer from MGE selected and incorporated at the leading edge of the CRISPR array
- protospacer selection requires a flanking protospacer adjacent motif
MATURATION;
- integration of the protospacer into the CRISPR array producers new spacers that are flanked byDIRECT REPEATS (DR) (20-50 nts long)
- transcription of the CRISPR array generates non coding RNA transcript (precursor-crRNA/ pre-crRNA)
- pre-crRNA processed by specific Cas proteins into mature crRNA. containing one spacer and at least 1 DR
INTERFERENCE;
- mature crRNAs bind to either single effector Cas proteins OR multiple Cas subunits to form surveillance RNP complex
- RNP complex recognises foreign MGEs that contain PAM sequence
- Cas nuclease activity promotes endonucleotlytic cleavage of the target strand/complementary strand to spacer crRNA

19
Q

in type II systems, what additional element is required for Cas9 activation ? describe this additional component ie where it is derived from, its functions, how it is assembled. Draw a diagram of the Cas9 assembly

A
  • tracRNA (trans-activating crRNA)
  • tracRNA found within intergenic regions of the CRISPR loci
  • small non coding RNA - 75-110 nt length
  • tracRNA contains sequence complementary to repeats of the crRNA and is required for crRNA processing and RNP assembly
  • complementary anti-repeat sequence base pairs to the repeat sequence of the crRNA (around 30nts in length) to form dsRNA duplex
  • RNase III cleaves the ds substrate to form individual crRNA:tracRNA:Cas9 complexes
    325 - 5 word
20
Q

draw a diagram of the full structure of the tracRNA : crRNA complex of Spcas9 system

A

word

21
Q

how long is the length of the double stranded duplex in the crRNA:tracRNA?

A

30nts

22
Q

how was the crRNA:tracRNA complex modified for use in research? how did this make using CRISPR systems easier?

A

linker joined on to create a chimeric single guide RNA

  • allows similutaneous expression of Cas9 and the sgRNA in cells (from either single or dual transfected plasmids)
  • OR can form the RNP complex in vitro (so the sgRNA (crRNA:tracRNA) and the Cas9) and transfect the WHOLE RNP
23
Q

how does the spCas9 system recognise its target?

A
  • conserved PAM sequence recognition by the Cas9. this PAM sequence needs to be adjacent to the complementary sequence
  • recognition of target complementary sequence by the gRNA
24
Q

what are the functions of the PAM sequence? what can happen if there is a single mutation?

A
  • allows differentiation between SELF and NON SELF genetic material
  • allows recognition of flanking complementary sequences to the gRNA
  • single mutation can prevent CRISPR-Cas cleavage
25
Q

what is the PAM sequence within spCas9 ?

A

5’ - NGG - 3’

26
Q

what is the result of there being many different PAM sequenceS?

A

targetted by number of different CRISPRcas variants

27
Q

describe the steps in PAM recognition

A
  • cas9 sgRNA searches for Target DNA by binding to the PAM site initially
  • flanking DNA then tested for complementarity to gRNA
  • Cas9 rapidly dissociates from regions that do not contain appropriate PAM site
  • time bound at PAM site dependent on amount of complementarity between gRNA and the flanking regions
28
Q

what structures within the Cas9 allow for specific recognition of the spCas9 PAM site? DAG

A
  • PAM DNA (NGG) binds in the +vely charged groove of Cas9
  • 5’ N not invovled in binding
  • 3’ GG recognised by2 Arg (R 1333,1335) residues within the PAM interacting motif (NUC domain) that H bond to the GG
  • PAM Cas9 interactions cause the destabilisation of adjacent DNA duplex pairing allowing the gRNA spacer strand to bind to Target DNA sequence
  • phosphate lock loop (Lys1107 and Ser1109) bind non specifically to the P backbone and stabilise the RNA:DNA hybridisation
  • 1st nucleotide of displaced NTS is stacked with the PAM duplex. the P makes a sharp kink causing a change in trajectory of the target strand
29
Q

what is the R loop ?

A

RNA:DNA heteroduplex and ssDNA of the NTS

- result of RNA strand invasion by Cas9 RNP complex

30
Q

give the steps in R loop formation. DAG of this overall R loop structure

A
  • recognition of PAM site causes adjacent DNA melting and unwinding in 3’-> 5’ direction
  • RNA strand invasion by gRNA forming RNA:DNA heteroduplex
31
Q

what is the accessible DNA within this R loop structure?

A
  • 9nt long of the ssDNA that remains accessible outside of the Cas9 protein (important in base editing)
32
Q

what are the results of seed region mismatches and PAM distal mutations?

A

SEED REGION MISMATCHES;

  • RNA strand invasion is non productive
  • Cas9 complex rapidly dissociates from the dsDNA

PAM DISTAL MUTATIONS;

  • RNA strand invasion still occurs
  • however less complementarity in distal sites altering R loop stability
  • DNA reannealing is favoured and cleavage rate decreased
33
Q

what is the main factor of target recognition and cleavage?

A

stable R loop formation
- this allows protein interactions from the Cas9 to be made all along the DNA:RNA heteroduplex which is required for recognition and cleavage

34
Q

where does the spacer DNA bind to the Target DNA within the Cas9?

A

+vely charged groove

35
Q

what is the length of the spacer sequence between the gRNA and the target strand? what are the mismatches it can tolerate?

A

overall length of spacer & Target DNA = 20nt

  • 17-18nt complementarity = still bind
  • 14-15nt complementarity = still bind however no cleavage = dead guide Cas9 with no nuclease activity
36
Q

give the 3 components of the NUC lobe

A

nuclease lobe

  • HNH domain. His-Asn-His motif nuclease
  • RuvC. RNase H like domain
  • PAM - interacting domain
37
Q

give the overall pathway of cleavage by the CRISPR RNP complex

A
  • binding of the substrate causing conformational change of the Cas9 to active conformation
  • HNH domain rotates 180degrees and places one of its active site residues (H840) to the target scissile P
  • HNH and RuvC now communicate causing simultaneous cleavage of the TS and NTS
  • after DNA substrate has been cleaved the the target strand remains bound to the gRNA for a certain amount of time. at this point, the RNP can no longer bind to additional targets
38
Q

describe the HNH domain

A
  • cleaves the target strand
  • one metal ion mechanism/catalyis to hydrolyse the scissile phosphates in the TARGET STRAND backbone
  • 3 AS catalytic residues (Asp 839, His840, Asn863)
  • Asp&Asn and Os from the scissile P cordinate an Mg2+
  • His coordinates H20 -> nucleophile to attack P
  • forming 5’ P and 3’ OH products (cleavage)
39
Q

describe the RuvC domain

A
  • cleaves the NON TARGET strand
  • 2 metal ion catalysis (Mn2+ )
  • 3 catalytic AS residues (Asp10, Glu762, His983, Asp986)
  • 3 of which coordinate the 2 Mn2+ ions (along with the O of the sccisle P). the other generates water nucleophile
  • cleavage

325 - World of RNA (5 decks)

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