methods 7

Question 1

how are protein affinity purification, pulldowns, and co-immunoprecipitation similar and different?

Answer 1

similar: methods to get protein out of a complex mixture using affinity of protein to a ligand/antibody

differences:
affinity purification uses an over expressed protein from a bacteria (so you may also purify bacterial interaction proteins), while pulldowns and co-ips use native cells.
co-ip proteins do not have a tag, while affinity purification and pulldowns downs do
co-ip is done with live cells that you fix in place to see what interactions naturally occur
the tagged bait protein in pulldowns in recombinant, while prey is not (or vice versa)

Question 2

What is the bait and what is the prey in yeast 3 hybrid?

Answer 2

it’s arbitrary, you can either use known RNA as the bait to grab a protein, or known protein to grab an RNA (you would still have make a hybrid RNA/hybrid protein so you need to know the potential interacting partners)

Question 3

why is it more common to use RNA as the bait in a yeast two hybrid?

Answer 3

there are fewer potential protein interaction partners you need to subclone hybrids for compared to the potential RNA interactors you would have to subclone hybrids for

Question 4

throughput of yeast two hybrid?

Answer 4

Low* to high

• One bait protein can be tested against a library of preys

Question 5

throughput of pull-down assay

Answer 5

low to medium

Question 6

throughput of co-immunoprecipitation assay

Answer 6

low to medium

Question 7

throughput of FRET

Answer 7

low*

• One bait protein can be tested against a library of preys

Question 8

throughput of bimolecular fluorecence complementation (BiFC) or split YFP

Answer 8

low*

• One bait protein can be tested against a library of preys

Question 9

what is RNAse I footprinting use to measure?

Answer 9

RNA protein interactions

Question 10

How does RNAse I footprinting work?

Answer 10

RNA is end labeled, incubated with protein of interest, fragemented with RNase I, resolved on gel and radiographed

Question 11

What does RNA immunoprecipitation measure

Answer 11

RNA-protein interactions

Question 12

how does RNA immunoprecipitation (RIP) work

Answer 12

RNA-protein complexes in lysate are incubated with protein beads (have an antibody against protein of interest or ligand if protein was tagged), beads isolated, RNA extracted, adaptor ligation, cDNA synthesis, PCR, next gen sequencing performed to id interaction partners

Question 13

how to measure weak protein-RNA interactions with RIP?

Answer 13

crosslink the RNA/protein complexes with UV before performing RNA immunoprecipitation

Question 14

What does a functional protein array measure/how?

Answer 14

A chip is covered in tiny droplets of recombinant proteins, screen interaction with a variety of labeled molecules (RNA, DNA, proteins, carbs, lipid, antibodies, etc) to detect interactions

Question 15

commonalities of genome editing tools

Answer 15

recognize target sequence

cut dsDNA in pre-designated location

trigger dsDNA repair machinery to either: re-join cut ends with NHEJ (small deletions/insertions) or HR (insert template DNA)

Question 16

what is a nuclease

Answer 16

an enzyme that cleaves nucleotides

Question 17

how many nucleotides does one zinc finger recognize?

Answer 17

3

Question 18

what happens when you attach a nuclease to a zinc finger

Answer 18

you have a zinc finger nuclease able to recognize a specific 3 bp sequence and cut dsDNA

Question 19

name of common nuclease combined with zinc fingers and TALENs

Answer 19

Fok1

Question 20

limitations of zinc finger nucleases

Answer 20

difficult to engineer

limited number of available zinc fingers

spacing the assemble correctly is difficult

Question 21

what does TALEN stand for

Answer 21

transcription activator-like effector nuclease

Question 22

components of a TALEN

Answer 22

Fok1 domain fused to a customizable DNA-binding domain

Question 23

What is a TAL effector (TALE) and how does it work

Answer 23

a protein able to bind DNA, recognizes DNA sequences by unique amino acid code in the middle of the protein. Variations in the amino acid code determine what bps it will bind

Question 24

what does CRISPR stand for

Answer 24

clustered regularly interspaced short palindromic repeats

Question 25

what does Cas stand for

Answer 25

CRISPR-associated

Question 26

explain how type II crispr system works

Answer 26

during initial viral infection: cas proteins recognize viral DNA @ the PAM sequence, chop up a protospacer (section of DNA), insert it into crispr array (an array of repeats of other chopped up DNA) as a “spacer” inbetween “crispr repeats”.

during subsequent viral infection: CRISPR loci (spacer) + part of crispr repeat are transcribed, transcripts processed to generate small CRISP RNAs (crRNA). crRNA hybridizes with tracrRNA (the crispr repeat is site of hybridization). This complex guides Cas9 endonuclease to invading DNA and recognizes it based on complementarity between spacer and target DNA, viral DNA is cleaved and degraded

Question 27

what is the PAM

Answer 27

protospacer adjacent motif, NGG

in viral genomes this sequence is always present next to the protospacer, in the bacteria crispr repeat this sequence is not present

makes sure cas9 only cuts up viral DNA and not DNA

Question 28

what does cas9 protein recognize?

Answer 28

DNA

Question 29

what does cas13 protein recognize?

Answer 29

RNA

Question 30

what is included in a vector to use for CRISPR?

Answer 30

2 oligos complimentary to desired target

cas9 protein sequence

gRNA

promoters

Question 31

what to do if no nGG sequence near a gene you want to edit with crispr?

Answer 31

pick a different PAM, there are many proteins aside from cas9 that recognize other PAM sequences

Question 32

what is the smallest cas protein?

Answer 32

cas 14, 400-700 amino acids in length

Question 33

what to do if you want to remove a large sequence of DNA?

Answer 33

use two chimeric gRNA and Cas9 proteins to cut from either end

Question 34

what is dCAS9? what can you use it for?

Answer 34

nuclease dead Cas9

use when you want to bind but not cut DNA, can attach an activation domain to activate a nearby gene, can attach an effector domain to make some chromatin or DNA mod, can add GFP to visualize gene

Question 35

Can you think of potential practical application of the RNA-targeting Cas13/gRNA?

Answer 35

fuse with a fluorescent protein and see where RNA is localized

RNA knockdown

modify RNA to study it

antiviral defense (target viral RNA)

Question 36

Why do you think the size of Cas proteins matters and what does Cas14 (~500aa) allow you to do experimentally that Cas9 or Cas12 (~1400aa) may not?

Answer 36

easier to transfect into smaller cells

viruses are small so cas12 wouldn’t fit

easier to build (shorter sequence to design)

bulky proteins can interfere with cell processes

less demanding of a cell to make smaller proteins

Question 37

Why do you think in genome editing making two dsDNA breaks with the help of two Cas9/gRNA complexes may be preferred over making a single dsDNA break with one Cas9/gRNA complex?

Answer 37

to knockout multiple genes/a region of genes

make two cuts, if one guide RNA is less efficient than the other and goal is just to knockout and you don’t know which is most efficient co-express many and hope any cuts your gene

80% of cut are repaired without indels