lecture 12 - genome editing using CRISP/Cas( Flashcards
genome editing
type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases
- enables specific targeting of sequencing within the genome without impacting the rest of the genome sequence
- potential to cure genetic diseases in patient specific manner
what is CRISPR/Cas9
clustered regulatory interspaced short palindromic repeats and CRISPR associated proteins
adaptive immune system of prokaryotes
three component system operated as a complex
Cas9
crRNA - crispr rns
tracrRNA
the complex cleaves invading DNA to prevent re infection by viruses
CRISPR acts as an immune system in prokaryotes against invading DNA/RNA
- invading DNA recognised and cut by Cas1-2 protein complexes into fragments called protospacers
- protospacers intergrated into CRISPR locus located into the bacterial genome
CRISPR acts as an immune system in prokaryotes against invading DNA/RNA
- invading DNA recognised and cut by Cas1-2 protein complexes into fragments called protospacers
- protospacers integrated into CRISPR locus located into the bacterial genome
the protospacers are transcribed to RNA and bind to cas9 protein
when invading strand re infects the complementary Cas9/RNA is recruited and Cas9
cuts DNA strands preventing infection
structure of CRISPR locus
transactivating RNA - sequence of RNA needed to bind to the guide RNA to allow cas 9 to bind to its target
then cas operon - encodes for cas protein components
indentical repeat array
spacer of invading DNA
the complex formed between the transactivating RNA and the protospacer/CRISPR RNA is called the guide RNA which
enables selective binding of Cas9 to invading DNA sequences
Cas9 is a single protein complex
Cas 1 and 2 has many proteins that forms the complex
tracrRNA and crRNA form
gRNA
protospacer adjacent motifs (PAM) in the invading DNA are required for cas9 to cleave DNA
PAM sequences are not present in the CRISPR locus provides a mechanism to distinguish bacterial self DNA from non-self DNA
expression of the protospacer sequences which are called gRNA when they come into contact with the transactivating RNA
Genome editing in the lab using CRISPR
we exploit the ability of CRISPR to specifically target and cleave DNA sequences to allow us to modify the genome of cells
linker loop
connects the crRNA and tracrRNA together
putting the cas9/gRNA complex at a desired locus of the genome will enable site specific cleavage through
nuclease activity
the gRNA should contain a protospacer sequence (target sequence) upstream of the
PAM site
once the DNA is cleaved the DNA repairs itself by
homology-directed repair
non-homologous end joining
homology directed repair only happens in
s phase
this is precise and there will be a sister chromatid to provide repair
non homologous end joining
happens everywhere else (primarily in interphase)
DNA repair by NHEJ is error prone
- introduces insertions or deletions into DNA
- impacts gene function
before its ligated
most changes result in premature stop codons can be used for a knock out mutation and normal gene product is not expressed
knock in by HDR (introduce a donor template that introduces a new change that we want)
instead of using the sister chromatid for the repair of the DNA we use the donor template we have inserted
remove PAM site so it cant be targeted by future cas9 proteins
androgen receptor signalling is a key driver of prostate cancer
current treatment aims to inactivate AR by blocking ligand binding
1. generate prostate cancer cell line expressing cas9 under the control of doxycycline
2. AR gene locus was targeted by CRISPS to create AR knock out cell line
could do knock in out stop codon in AR gene
