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