Mechanisms of CRISPR-Cas
Mature crRNAs bind to single ___ proteins or multiple ___ subunits to form a:
Cas, Cas, RNP complex
pre-crRNA are processed by specific ___ proteins to produce short __________ that contain a single _______ and at least 1 _________
Cas, crRNAs, spacer, repeat
CRISPR type II is characterised by:
Cas9
Selected PAM sequence is dependent on the species of _____
Cas9
The Scaffold of the gRNA is for:
Cas9 binding
CRISPR stands for
Clustered Regularly Interspaced Short Palindromic Repeats
The RNP complex recognises:
Foreign DNA containing a PAM sequence
The NUC lobe of Cas9 contains the following three domains:
HNH nuclease domain, RuvC-like nuclease domain, PAM-interacting domain
Substrate binding causes a rotation of the ___ domain towards the target strand, which is necessary for activation of ___________ activity
HNH, nuclease
CRISPR Type __ is a bacterial _________ system that has been modified for genome engineering
II, immune
Cas9 is composed of a ________________ lobe and a _________________ lobe
Nuclease (NUC), Recognition (REC)
The HNH uses _______________ as a cofactor to hydrolyse ____________
One metal ion, phosphates
Protospacer selection requires flanking _____ sequence
PAM
The gRNA spacer must be present immediately upstream of a:
PAM
In interference, DNA cleavage occurs three bases after the:
PAM site
Cas9-sgRNA search for target DNA by binding to ____ sequence and then testing the flanking DNA for potential complementarity to the ______________
PAM, Guide RNA
Mismatches in the seed region causes rapid dissociation, but mutations in the ________________ region allow productive RNA strand invasion, but will alter _________ stability which favours DNA re-annealing
PAM-distal, R-loop
When Cas9 has found a target with the appropriate PAM, it triggers DNA unwinding from the _______________ end to the __________________ end
PAM-proximal, PAM-distal
PAM DNA binds in ___________________________________ on Cas9
Positively charged groove
PAM stands for:
Protospacer Adjacent Motif (PAM)
Target recognition and cleavage depends on stable ___________ formation
R-Loop
Class two systems involve ____-guided effector complexes that require a single ______________ protein for interference
RNA, multidomain
Co-ordinated action of the HNH and ______ nucleases hydrolyses phosphates to create a _____________________
RuvC-like, blunt end dsDNA break
Guide RNA is a synthetic RNA containing a _____________ sequence and a __________________
Scaffold, Spacer
The gRNA spacer must be unique compared to:
The rest of the genome
Cas nuclease activity destroys sequences complementary to:
The spacer segment
tracrRNA stands for:
Trans activated CRISPR RNA
In Interference, after cleavage, the target strand remains base paired to _____ and the protein is unable to:
gRNA, bind additional targets
gRNA spacer defines the region of the _________ to be targeted
genome
gRNA binding to Cas9 changes conformation from ___________________________ to ___________________
non-DNA binding, DNA-binding
RuvC-like domain cleaves the _____________ strand
non-target
tracrRNA contains sequences complementary to repeat sequences in ____________
pre-crRNA
tracrRNA forms double stranded RNA with ________ which prompts cleavage by ______________ to form small hybrid molecules that bind to _________
pre-crRNA, RNase III, Cas9
Transcription of the CRISPR array creates a non-coding RNA known as a:
precursor CRISPR RNA(pre-crRNA)
Integration of protospacer into the host CRISPR array produces new ___________ flanked by 20-50 ________________________
spacers, direct repeats
The HNH domain cleaves the _________ strand
target
RuvC-like domain uses __________________ as a cofactor to hydrolyse _____________
two metal ions, phosphates
gRNA spacer is about ___ nucleotides long
20
SpCas9 recognises 5'-NGG-3', ___ does not interact with Cas9 but ____ forms H-bonds with a pair of ____ in the beta hairpin PAM interacting motif
5'-N, GG-3', Args
For S. pyogenes the PAM sequence is:
5'-nGG-3'
PAM sequences distinguish self from non-self and single mutations are often sufficient to prevent _____________________
CRISPR-Cas cleavage
Cas9 rapidly ____________ from DNA that does not contain the appropriate PAM sequence
dissociates