5 - Mechanisms of CRISPR Cas - Bose Flashcards
(39 cards)
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
