Crispr Cas Flashcards
Crispr cas in humans is used for genome editing what does this mean
Inserting deleting or altering genes using nucleases
What are cas
Crispr associated proteins
Why is crispr cas found in 40% of bacterial genomes
For adaptive immunity against viral infections
What 3 component complex makes the crispr cas bacteria system
Cas 9 protein nuclease
Cr rna (protospacers)
Tracr rna
What cleaves invading viral dna into protospacers (cr rna)
Cas 1 and 2
What happens once protospacers cr rna are made from cas 1 and 2 viral cleavage
Incorporated into the crispr locus
Why are protospacers incorporated to crispr locus in bacteria
If reinfection via that same virus occurs, protospacers can guide and bind to cas 9 to the viral dna complementary/ homologous to protospacers cr rna
Cas 9 then cleave phage dna
What makes up the crispr locus
Tracr rna
Cas operon (for diff types of cas proteins)
Protospacers cr rna
In between cr rna there are repeat arrays
What Hetero dimerises to form guide rna
Tracr rna and cr rna protospacers
What is the diff between type 1/ or 3 cas and type 2
1 and 3 types are multisubunit
Type 2 is multi domain
What type of cas is 2 and 3
A type 1 and 3 multisubunit cas
What type of cas is 9
Type 2 multi domain
Which domain of cas 9 binds g rna
Rec 1 and 2
Which lobe of cas 9 is responsible for cleaving comp dna, non comp dna , and Pam interaction
NUC lobe
What is Pam and why is it important
Sequence on invading dna upstream or down of the protospacer dna comp sequence which allows cas9 to detect self bacterial dna and non self dna
Does cas 9 cleave invading dna if they am site is not near to grna binding sequence
No. Cas 9 needs Pam to cleave
Is pam found in crispr locus
No
How have tracr rna and cr rna been simplified into 1 g rna for genome editing
Linkage through a linker loop
What does deposition of a cas 9 g rna cause on a specific target site
Gene knock in or out because of the repair system
How are cas 9 and g rna up regulated / expressed for genome eidditjng
in vitro plasmid with cas 9 and g rnawhich gets transacted into euk cell for expression
How is grna/cas9 made specific for genome editing
G rna is comp to 1 of the dna strands in a specific target gene for example. The dna has a Pam sequence adjacent to the protospacer sequence target
What causes repair system stimulation
Double strand break via nucleases in cas
Which repair pathway causes a gene knockout due to it being error prone
Non homologous end joining NHEJ
How does NHEJ cause gene knockouts by degrading mrna
Addition of nucleotides by terminal transferases on the dna breaks causes insertion or deletion. Insertion can cause PTCs
PTCs mean exon junction complexes EJCs won’t be knocked off by ribosomes
Recruit upf proteins for degradation of rna (NMD)
Which type of repair system causes gene knock in
Homology directed repair
How does homology directed repair work
The cut gene target by cas9 undergoes homologous recombination with a sister chromatid of desired target gene
Because the cut site on dna has homology arms to the sister chromatid which will exchange the gene of interest forming knock in
What phase do chromatids need to be in for homology directed repair HDR
S phase
How is crispr cas delivered ex vivo and in vivo
Ex vivo via removal of cells then put back in
In vivo putting cas crispr delivered into organ
How can crispr be used to treat hiv
Cause gene knockout in ccr5 by inducing a mutation via nhej for example
