3 - gene editing drugs Flashcards
what is genomics
the branch of biology dealing with aspects of the genome (all genetic info in an organism)
is genomics descriptive
yes
what is predictive modelling
predicting things like pharmacogenetics and susceptibility to disease
how do you do gene sequencing (2)
genome wide association studies and classification of organisms
what is pharmacogenetics
use genes to see how you will be affected by drugs
what is genome wide association study
compare two big populations, one with gene and one without
what are monogenic diseases
diseases caused by a mutation in a single gene
what are more monogenenic diseases (gain vs loss of function)
loss of function
is it easier to find small molecule activators in inhibitors
hard to find hinhibitors, harder to find activators
what are 3 drawbacks to gene editing
off-target effects, mutations, irreversibility
what do Zinc fingers bind to
specific DNA triplet sequences
what are most zinc fingers part of
transcription factors
can all triplets be recognized by natural zinc fingers?
no (so we engineered new ones)
how many zinc fingers would be used to target a 9 bp sequence
3
what is a zinc finger nuclease
two zinc finger arrays linked to subunits of the heterodimeric Fok1 endonuclease
what must the heterodimeric Fok1 endonucleases have to work
both of the 2 subunits to come together
what do endonucleases do
cut DNA internally
is heterodimeric Fok1 endonucleases specific to where it cuts?
no, so you attach them to zinc fingers to guide them
where do the zinc finger nucleases bind
to the left and right sequences surrounding the spacer where the cut occurs
where does a TALE domain bind
to 1bp of DNA
how do TALEs target long sequences of DNA
they can be linked
how is it determined where TALEs bind
two amino acids determine which specific base is bound (all the other amino acids are constant, just 2 variants)
what is TALEN
two tale arrays linked to subunits of the heterodimeric Fok1 endonuclease
is protein engineering required for TALENs
NOOOO (each base can be targeted by a particular domain)
what does non-homologous end joining to do genes
insertions or deletions that knockout / inactivation a gene
what are 2 DNA repair mechanisms
non homologous end joining and homologous recombination
what does homologous recombination to do genes
knock-in or gene editing by supplying repair DNA
how does non homologous end joining often cause a knockout of genes
cut makes staggered overhands, they get degraded(remove stuff) then fused together then boom deletion
how can homologous recombination be used to make a new gene
the DNA is cut then resupplied with new template DNA
what is CRISPR in prokaryotes part of
their prokaryotic immune system that defends against viruses
what are the 4 steps in CRISPR immune stuff in viruses
1-invasion
2-adaptation
3-production
4-targeting
what happens in invasion of CRISPR
the virus gets into the bacterial cell
what happens in adaptation stage of CRISPR
DNA from bacteriophage is stored into the CRISPR sequence
what happens in production stage of CRISPR
CRISPR RNA is made from the memory locus that is complementary to the bacteriophage
what happens in targeting stage of CRISPR
endonuclease Cas9 binds to RNA to find the homologous sequence on the DNA of the bacteriophage then kills it
what is the difference with Cas9 and CRISPR
they the same
what are 3 main components of CASRP Cas9
Cas9 endonuclease,
crRNA with 20bp sequence used to target, trans-activating crRNA
what is the PAM sequence
NGG
Cas9 has a specificity for it
what is the first step of CRISPR Cas9 cleavage
Cas9 binds to PAM
what happens after Cas9 binds to PAM
cas9 tries to pair guide RNA with DNA
what are the two nuclease domains of cas9
RuvC and HNH
what happens after cas9 pairs guide RNA with DNA
the two nuclease domains (RuvC and HNH) cut the DNA to have a double-stranded break
what happens after the RuvC and HNH cut the DNA
non homologous end joining or homologous recombination come to fix it
what are the 3 important steps in determining the 20-bp sequence to target
1-identify PAM (NGG) sequence in DNA you want to target
2-count 20 nucleotides upstream PAM to find 5’ start of sgRNA target sequence
3- determine the actual sgRNA sequence
what is sgRNA
CRISPR RNA
what is PAM sequence
NGG
why cant cas9 get into cells easily
they are large and complex and impermeable to the cell membrane
what are 3 ways you can get gene editing tools into cells
1-viruses
2-physical disruption of membrane (small needle)
3-chemical coating(attach to nanoparticle to get it through the membrane)
what could happen if you cleave DNA
induce DNA-damage response in cell, causing cell death
what does ex-vivo mean
therapy is performed outside the body
what is in vivo
when therapy is treated directly in the body
what is somatic cell
cell that is not reproductive and not passed on
what is germ-line cell
cell that is reproductive and passed on
what is the sequence of events in Cas9 DNA cleavage reaction *3)
PAM binding
crDNA hybridization
activation of nucleases
