M3 L16: Fwd and rev genetic screens

Question 1

2 questions for understanding the genetic basis of phenotypic var

Answer 1

1) how do we det a gene’s function?

2) how do we det if a gene is responsible for a trait

Question 2

2 ways to answer the 2 questions

Answer 2

1) fwd genetic screen: start w/ phen in mind, mutagenize identical starting organisms, look for phen of interest, identify causal gene, infer gene fxn

2) rev genetic screen: start w/ known gene, mutagenize the sequence, obs phen changes, infer gene fxn

Question 3

who did the first fwd genetic screens?

Answer 3

hermann muller

discovered can mutate drosophila by exposing to radiation

Question 4

example of fwd genetic screen

Answer 4

WT drosophila can learn to associate a scent w/ an electric shock –> take action if they experience same smell again

mutants cannot learn or cannot remember

Benzer and Quinn looked for flies that couldn’t associate –> identified TF creb

Question 5

why are fwd genetic screens unbiased?

Answer 5

don’t need prior knowledge of the gene

Question 6

general design of a fwd genetic screen

Answer 6

start with identical organisms –> mutagenize

look for phenotypes (visually or by plating) –> isolate mutant

Question 7

specific considerations for fwd genetic screens

Answer 7

saturation mutagenesis is ideal: mutate each gene in the genome at least once

choice of phenotype influences 3 other choices:
1) organism
2) mutagen
3) strategy for identifying dom/rec muts

Question 8

key pts for choosing an organism

Answer 8

can only do screens in orgs that reproduce in large #s quickly

org needs to be crossable

starting orgs need to be genetically identical

use simplest org possible

Question 9

key pts for choosing mutagen

Answer 9

depends on organism and type of mutations desired

EMS/UVC –> point muts

high energy rad –> rearrangements

transposon mutagenesis –> null alleles

Question 10

what are the 3 strategies for obs dom/rec muts

Answer 10

F1 screen: see dom muts immediately in diploid yeast

F2 screen: see rec muts in orgs that can self (self a heterozygote –> 3:1 ratio dom:rec)

F3 screen: see rec muts in orgs that cannot self (cross F1 heterozygote to parent –> F2 has same mut as F1, cross F2 and F1 –> 25% F3s homo rec mut)

Question 11

how to identify recessive lethals on the x chrom? who discovered the technique?

Answer 11

CLB screen strategy, Muller

Question 12

what’s a CLB chrom? what are the components?

Answer 12

balancer chromosome

C: crossover suppression bc of inversion

L: recessive lethal

B: bar eyes mutation (dominant) –> mut present means indiv has CLB chrom

Question 13

what are the crosses and outcomes for CLB screen

Answer 13

1) cross mutagenized male with CLB female –> isolate the CLB female F1s (have mutant X from male)

2) cross CLB F1 females to WT males –> male F2s get CLB X or mutant X

2:1 female:male –> X mut nonlethal (CLB males die, X mut males live)

1:0 female: male –> X mut and CLB males all die

Question 14

pro and con for using haploids for genetic screens

Answer 14

dom and rec muts obs immediately

but cannot obs effect of LOF mut in essential genes (die)

Question 15

how to obs effect of LOF mut in essential genes?

Answer 15

conditional mutants where the mut phen is only expressed in certain conditions –> obs phens in continuous range of conditions

Question 16

example of a conditional allele/mut

Answer 16

temp sensistive muts (cdc mutants) –> mut in cyclins for controlling cell cycle

only yeast cells in high temp die –> LOF (probably missense) mut decreases protein stability at high temp

dif cdc genes correspond to dif points in cell cycle –> obs what stage yeast cell gets stuck in at dif temps

Question 17

how to identify secondary muts that modify the mutant phen? what are the types of screens?

Answer 17

modifier screens (mutagenize a mutant)

1) enhancer screens: more severe mut phen

2) suppressor screens: muts that restore WT

experimental evolution

Question 18

examples of modifier screens

Answer 18

Su(var) and E(var): mutagenize flies with variegated eyes –> see if eyes are more red or more white

Question 19

what’s experimental evolution

Answer 19

impose strong selective pressures for a certain phenotype

grow orgs with muts for slow growth in rich media (selective pressure for a supressor mut to restore WT phen) –> can grow much faster than everyone else

Question 20

what is synthetic lethality

Answer 20

most extreme type of interacting mutation

two muts separately are not lethal, but if both present in one indiv –> lethal

Question 21

who discovered synthetic lethality? what phenotypes?

Answer 21

alfred sturtevant
X linked pn and K=pn muts (prune and killer prune)

cross female with pn and make with K-pn –> all males die –> K-pn only lethal if pn also present

Question 22

2 poss explanations of synthetic lethality

Answer 22

1) genetic redundancy/parallel complementary pathways: 2+ genes do the same function –> viable as long as you have 1 WT copy (all the rest can be NULL)

2) within pathway interactions: 2+ genes do dif functions in same pathway –> can survive w/ LOF in one gene for the pathway (but not both and NONE CAN BE NULL)

Question 23

how to identify causal mutations via sequencing

Answer 23

sequence all indiv with that phenotype –> look for common mut that each indiv has (very unlikely for all indiv w/ that phen to have the mut and for it to not be causal)

Question 24

2 drawbacks of fwd genetic screens

Answer 24

1) biased mut spectrum: mutagens can only give rise to certain types of muts

2) questionable ecological relevance of muts: may be able to produce a mut in vitro but has dif effect in vivo (due to antagonistic pleiotropy where mut changes two phenotypes –> increase and decrease fitness)

Question 25

how can you avoid the two drawbacks of fwd genetic screens

Answer 25

experimental evo

wide range of natural muts

select against muts that decrease fitness

Question 26

why do reverse genetics?

Answer 26

genomes are redundant so one mutant phen could be from muts in several dif genes

Question 27

how to make precise genetic edits? why does it work?

Answer 27

crispr-cas9 –> use guide RNA to make double stranded breaks at spec seqs

repair via NHEJ –> small indels

repair via SDSA –> single nucleotide changes using template with desired mut

Question 28

where did crispr come from

Answer 28

salt marshes off spain coast

in archaean haloferax mediterranei

genome has identical repeats with nonidentical seqs in between

identical seqs = clustered regularly interspersed palindromic repeats

adjacent genes = crispr associated genes –> encode DNA endonuclease as a complex or one protein

Question 29

how does crispr work in bac and arch

Answer 29

defense against invading nucleic acids

spacers = crispr seqs derived from past phage infection

Cas proteins use crispr spacers as guides

crispr seqs –> noncoding RNAs (crRNAs w/ unique spacer seq and identical repeat seq)

tracrRNA made from gene upstream crispr locus (tracrRNA is partially complementary to repeat seqs and forms stem loop that binds cas endonuclease –> bind crRNA to cas9)

cas finds and cuts complementary seqs

Question 30

can crispr arrays be passed on to offspring

Answer 30

yes

Question 31

how to apply crispr-cas9 for science

Answer 31

replace crRNA with seq complementary to a seq of interest

simplify tracrRNA and crRNA into single molecule (guideRNA/gRNA) and use Cas9 (single protein w/ cas endonuclese fxn)

Question 32

why is Cas9 highly specific? can we still get off target effects?

Answer 32

single mismatch is usually enough to prevent Cas9 from cutting but genomes are redundant so can cut at wrong copy of right sequence

Question 33

applications of crispr-Cas9

Answer 33

1) gene tharapy: edit gene back to WT

2) agriculture: make hornless cattle

3) evolution: resurrect extinct species by placing their genes into extant organisms

4) public health: implement artificial gene drive sys –> make mosquitos extinct

Question 34

implication of RNAi for reverse genetics

Answer 34

can knock down any gene of interest (destroy mRNA or prevent its translation)

inject dsRNA complementary to the gene you want to knock out –> processed by dicer –> associate w/ RISC –> RISC degrades mRNAs

Question 35

how to achieve heritable gene knockdown

Answer 35

insert miRNA transgene into org’s genome

Question 36

what’s a good organism for RNAi? why?

Answer 36

c. elegane (eat E. coli)

can manipulate E. coli genome to express dsRNA complementary to c. elegans gene

can have transgenerational effect bc c. elegans has RNA dep RNA polymerase –> start making the dsRNA from E. coli

Question 37

what are chimeric genes?

Answer 37

genes in a different context - under a different gene’s regulatory sequences –> ectopic expression

Question 38

example of chimeric gene? what does the result tell us?

Answer 38

chimeric eyeless allele in flies –> eyeless expressed in all imaginal discs –> dev anatomical eyes in wrong places

tells us about gene fxn when expressed in wrong context, but not about the gene’s normal function

early eyeless expression –> lethal, but purpose eyeless is not to kill embryo

ectopic eyes tell us
1) cells in imaginal discs can differentiate into dif structures

2) eyeless sufficient to drive eye dev in other imaginal discs that don’t usually become eyes

Question 39

Is fwd or rev genetics more amenable to random mutagenesis and crispr-cas9? explain

Answer 39

Forward is more amenable to random mutagenesis because it’s unbiased - you don’t need any prior knowledge of the sequence. If the mutations are random, you have no way of knowing what sequence they’re in, so you can’t do reverse genetics.

Reverse genetics is more amenable to CRISPR-Cas9 because this mechanism allows you to induce indels or single nucleotide mutations at very specific sequences. You would need to know the sequence beforehand.

Question 40

Design a forward genetic screen for point mutations that cause cadmium resistance. Choose an organism, a mutagen, and a method to isolate both dominant and recessive mutations. Justify each of your choices.

Answer 40

Organism: haploid yeast – has genes for heavy metal resistance, reproduces quickly

Mutagen: radiation – causes chromosomal rearrangements → putting genes under different promoters can increase expression

Isolation: place all F1 offspring in media with high concentrations of cadmium → dominant and recessive phenotypes will both be expressed (any yeast that survive in high cadmium concentrations) bc yeast is in haploid state

Question 41

Design a forward genetic screen for point mutations that cause increases in susceptibility to heat induced seizures. Choose an organism, a mutagen, and a method to isolate both dominant and recessive mutations. Justify each of your choices.
Organism: Mice (unless drosophila can have seizures, then use them bc they’re simpler) – relatively simple but can’t have anything too simple bc it must be able to have seizures

Mutagen: Chemical like EMS or UV radiation → causes point mutations

Isolation: must do F3 screen because mice cannot self (Mutagenize male mouse gametes, cross to WT female → 50% of F1 offspring are mutated and all have different mutations → cross each to WT parent → 50% of each strain of F2s are mutated and within each strain, all offspring have the same mutation → cross to each other → 25% F3 offspring will be homozygous recessive (should have more seizures in heated environments).

Answer 41

Design a forward genetic screen for point mutations that cause increases in susceptibility to heat induced seizures. Choose an organism, a mutagen, and a method to isolate both dominant and recessive mutations. Justify each of your choices.
Organism: Mice (unless drosophila can have seizures, then use them bc they’re simpler) – relatively simple but can’t have anything too simple bc it must be able to have seizures

Mutagen: Chemical like EMS or UV radiation → causes point mutations

Isolation: must do F3 screen because mice cannot self (Mutagenize male mouse gametes, cross to WT female → 50% of F1 offspring are mutated and all have different mutations → cross each to WT parent → 50% of each strain of F2s are mutated and within each strain, all offspring have the same mutation → cross to each other → 25% F3 offspring will be homozygous recessive (should have more seizures in heated environments).

Question 42

Why are F3 screens needed for identifying recessive mutations in organisms that cannot self but these mutations can be identified in F2 screens in organisms that can self?

Answer 42

When you mutagenize male gametes, different gametes will have different mutations → 50% offspring are mutated and all have different mutations. To express the recessive phenotype, you need two copies of the mutation, and the only ways to get that are to cross a mutant F1 with itself → 25% of the F2s are homozygous

OR, if the organism cannot self, you have to mutagenize the male gametes, cross with a normal female → again 50% F1 are mutants and each have a different mutation → cross one mutant F1 with a WT parent → 50% of the F2s are mutants but all have the same mutation → can cross original F1 with an F2 → 25% homozygous for a mutation.

Question 43

In a forward genetic screen for S. cerevisiae mutants that can grow in elevated concentrations of azole drugs, you identify a mutant that has moderate levels of resistance but also a severe growth defect in rich media. How would you go about identifying a secondary mutation that enhances the resistance phenotype? How would you go about identifying a mutation that rescued wild type growth in rich media?

Answer 43

Identify a secondary mutation that enhances the resistance phenotype by mutagenizing the mutant → plate in higher concentration of azole drug, observe change in phenotype, identify gene, and interpret function (do a forward genetic screen)

Identify a mutation that restores WT in rich media by doing experimental evolution. Grow the yeast mutants in rich media → whichever cell can mutate again and restore WT function will have a huge advantage and will be able to grow and reproduce much faster than the rest of the cells. Identify the gene that was mutated the second time and infer its function.