5 - Mechanisms of CRISPR Cas - Bose Flashcards
what does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
what is the type and class of CRISPR system used in molecular biology? briefly describe this system
Class 2, type II CRISPR Cas9
- bacterial immune system that has been modified for genome engineering
CRISPR Cas is highly dependent upon the ____ and ____ of non coding RNA transcripts to create an ___ complex
expression
processing
RNP
why is CRISPR such a useful tool in molecular biology?
highly adaptable, countless variations of system for different uses
what are the 2 components of the CRISPR system? briefly describe them
- 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
what is the prerequisite for the spacer target sequence?
immediately upstream of a PAM site
draw a simple diagram of the CRISPR Cas components and recognition of DNA
325 -5 word
what is the PAM site?
- protospacer adjacent motif
- present on the non target strand
- PAM specific to the species of Cas present
- eg spCas9 has PAM sequence NGG
where does the DSB occur on the DNA?
3nt upstream of the PAM site
describe the architecture of Cas9
- include the names of the lobes, where the spacer and scaffold sequences bind etc
DAG
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)
describe how Cas9 specificity to the target arises?
- Cas9 binding to the PAM site
- complementarity and base pairing between the gRNA spacer and the target strand
what are the ways of repairing this Cas9-induced DSBs? how is this useful?
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
how are Class 2 CRISPR systems characterised? name one type of this and name the Cas protein invovled
- 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
what is CRISPR Cas immunity?
- bacteria and archae, Crispr Cas immunity provides defence against mobile genetic elements (MGEs) from phage infection
how has CRISPR evovled in bacteria?
ongoing competition between phages/MGEs and bacteria have created rapid evolution and diversification of the CRISPR loci
what does the CRISPR system look like in the genome? what are the components and what do they code for
- CRISPR array (encoding noncoding RNA)
- neighbouring Cas genes (encoding protein)
draw a diagram showing the OVERALL scheme of the CRISPR system in bacteria. In the diagram include an infecting bacteriophage
325 - 5 word
name and describe the 3 steps in the CRISPR pathway and defence against MGEs
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
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
- 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
draw a diagram of the full structure of the tracRNA : crRNA complex of Spcas9 system
word
how long is the length of the double stranded duplex in the crRNA:tracRNA?
30nts
how was the crRNA:tracRNA complex modified for use in research? how did this make using CRISPR systems easier?
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
how does the spCas9 system recognise its target?
- 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
what are the functions of the PAM sequence? what can happen if there is a single mutation?
- 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
what is the PAM sequence within spCas9 ?
5’ - NGG - 3’
what is the result of there being many different PAM sequenceS?
targetted by number of different CRISPRcas variants
describe the steps in PAM recognition
- 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
what structures within the Cas9 allow for specific recognition of the spCas9 PAM site? DAG
- 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
what is the R loop ?
RNA:DNA heteroduplex and ssDNA of the NTS
- result of RNA strand invasion by Cas9 RNP complex
give the steps in R loop formation. DAG of this overall R loop structure
- recognition of PAM site causes adjacent DNA melting and unwinding in 3’-> 5’ direction
- RNA strand invasion by gRNA forming RNA:DNA heteroduplex
what is the accessible DNA within this R loop structure?
- 9nt long of the ssDNA that remains accessible outside of the Cas9 protein (important in base editing)
what are the results of seed region mismatches and PAM distal mutations?
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
what is the main factor of target recognition and cleavage?
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
where does the spacer DNA bind to the Target DNA within the Cas9?
+vely charged groove
what is the length of the spacer sequence between the gRNA and the target strand? what are the mismatches it can tolerate?
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
give the 3 components of the NUC lobe
nuclease lobe
- HNH domain. His-Asn-His motif nuclease
- RuvC. RNase H like domain
- PAM - interacting domain
give the overall pathway of cleavage by the CRISPR RNP complex
- 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
describe the HNH domain
- 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)
describe the RuvC domain
- 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
