3 - gene editing drugs

Question 1

what is genomics

Answer 1

the branch of biology dealing with aspects of the genome (all genetic info in an organism)

Question 2

is genomics descriptive

Answer 2

yes

Question 3

what is predictive modelling

Answer 3

predicting things like pharmacogenetics and susceptibility to disease

Question 4

how do you do gene sequencing (2)

Answer 4

genome wide association studies and classification of organisms

Question 5

what is pharmacogenetics

Answer 5

use genes to see how you will be affected by drugs

Question 6

what is genome wide association study

Answer 6

compare two big populations, one with gene and one without

Question 7

what are monogenic diseases

Answer 7

diseases caused by a mutation in a single gene

Question 8

what are more monogenenic diseases (gain vs loss of function)

Answer 8

loss of function

Question 9

is it easier to find small molecule activators in inhibitors

Answer 9

hard to find hinhibitors, harder to find activators

Question 10

what are 3 drawbacks to gene editing

Answer 10

off-target effects, mutations, irreversibility

Question 11

what do Zinc fingers bind to

Answer 11

specific DNA triplet sequences

Question 12

what are most zinc fingers part of

Answer 12

transcription factors

Question 13

can all triplets be recognized by natural zinc fingers?

Answer 13

no (so we engineered new ones)

Question 14

how many zinc fingers would be used to target a 9 bp sequence

Answer 14

3

Question 15

what is a zinc finger nuclease

Answer 15

two zinc finger arrays linked to subunits of the heterodimeric Fok1 endonuclease

Question 16

what must the heterodimeric Fok1 endonucleases have to work

Answer 16

both of the 2 subunits to come together

Question 17

what do endonucleases do

Answer 17

cut DNA internally

Question 18

is heterodimeric Fok1 endonucleases specific to where it cuts?

Answer 18

no, so you attach them to zinc fingers to guide them

Question 19

where do the zinc finger nucleases bind

Answer 19

to the left and right sequences surrounding the spacer where the cut occurs

Question 20

where does a TALE domain bind

Answer 20

to 1bp of DNA

Question 21

how do TALEs target long sequences of DNA

Answer 21

they can be linked

Question 22

how is it determined where TALEs bind

Answer 22

two amino acids determine which specific base is bound (all the other amino acids are constant, just 2 variants)

Question 23

what is TALEN

Answer 23

two tale arrays linked to subunits of the heterodimeric Fok1 endonuclease

Question 24

is protein engineering required for TALENs

Answer 24

NOOOO (each base can be targeted by a particular domain)

Question 25

what does non-homologous end joining to do genes

Answer 25

insertions or deletions that knockout / inactivation a gene

Question 26

what are 2 DNA repair mechanisms

Answer 26

non homologous end joining and homologous recombination

Question 27

what does homologous recombination to do genes

Answer 27

knock-in or gene editing by supplying repair DNA

Question 28

how does non homologous end joining often cause a knockout of genes

Answer 28

cut makes staggered overhands, they get degraded(remove stuff) then fused together then boom deletion

Question 29

how can homologous recombination be used to make a new gene

Answer 29

the DNA is cut then resupplied with new template DNA

Question 30

what is CRISPR in prokaryotes part of

Answer 30

their prokaryotic immune system that defends against viruses

Question 31

what are the 4 steps in CRISPR immune stuff in viruses

Answer 31

1-invasion 2-adaptation 3-production 4-targeting

Question 32

what happens in invasion of CRISPR

Answer 32

the virus gets into the bacterial cell

Question 33

what happens in adaptation stage of CRISPR

Answer 33

DNA from bacteriophage is stored into the CRISPR sequence

Question 34

what happens in production stage of CRISPR

Answer 34

CRISPR RNA is made from the memory locus that is complementary to the bacteriophage

Question 35

what happens in targeting stage of CRISPR

Answer 35

endonuclease Cas9 binds to RNA to find the homologous sequence on the DNA of the bacteriophage then kills it

Question 36

what is the difference with Cas9 and CRISPR

Answer 36

they the same

Question 37

what are 3 main components of CASRP Cas9

Answer 37

Cas9 endonuclease, | crRNA with 20bp sequence used to target, trans-activating crRNA

Question 38

what is the PAM sequence

Answer 38

NGG | Cas9 has a specificity for it

Question 39

what is the first step of CRISPR Cas9 cleavage

Answer 39

Cas9 binds to PAM

Question 40

what happens after Cas9 binds to PAM

Answer 40

cas9 tries to pair guide RNA with DNA

Question 41

what are the two nuclease domains of cas9

Answer 41

RuvC and HNH

Question 42

what happens after cas9 pairs guide RNA with DNA

Answer 42

the two nuclease domains (RuvC and HNH) cut the DNA to have a double-stranded break

Question 43

what happens after the RuvC and HNH cut the DNA

Answer 43

non homologous end joining or homologous recombination come to fix it

Question 44

what are the 3 important steps in determining the 20-bp sequence to target

Answer 44

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

Question 45

what is sgRNA

Answer 45

CRISPR RNA

Question 46

what is PAM sequence

Answer 46

NGG

Question 47

why cant cas9 get into cells easily

Answer 47

they are large and complex and impermeable to the cell membrane

Question 48

what are 3 ways you can get gene editing tools into cells

Answer 48

1-viruses 2-physical disruption of membrane (small needle) 3-chemical coating(attach to nanoparticle to get it through the membrane)

Question 49

what could happen if you cleave DNA

Answer 49

induce DNA-damage response in cell, causing cell death

Question 50

what does ex-vivo mean

Answer 50

therapy is performed outside the body

Question 51

what is in vivo

Answer 51

when therapy is treated directly in the body

Question 52

what is somatic cell

Answer 52

cell that is not reproductive and not passed on

Question 53

what is germ-line cell

Answer 53

cell that is reproductive and passed on

Question 54

what is the sequence of events in Cas9 DNA cleavage reaction *3)

Answer 54

PAM binding crDNA hybridization activation of nucleases