M3 L16: Fwd and rev genetic screens Flashcards
2 questions for understanding the genetic basis of phenotypic var
1) how do we det a gene’s function?
2) how do we det if a gene is responsible for a trait
2 ways to answer the 2 questions
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
who did the first fwd genetic screens?
hermann muller
discovered can mutate drosophila by exposing to radiation
example of fwd genetic screen
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
why are fwd genetic screens unbiased?
don’t need prior knowledge of the gene
general design of a fwd genetic screen
start with identical organisms –> mutagenize
look for phenotypes (visually or by plating) –> isolate mutant
specific considerations for fwd genetic screens
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
key pts for choosing an organism
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
key pts for choosing mutagen
depends on organism and type of mutations desired
EMS/UVC –> point muts
high energy rad –> rearrangements
transposon mutagenesis –> null alleles
what are the 3 strategies for obs dom/rec muts
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)
how to identify recessive lethals on the x chrom? who discovered the technique?
CLB screen strategy, Muller
what’s a CLB chrom? what are the components?
balancer chromosome
C: crossover suppression bc of inversion
L: recessive lethal
B: bar eyes mutation (dominant) –> mut present means indiv has CLB chrom
what are the crosses and outcomes for CLB screen
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
pro and con for using haploids for genetic screens
dom and rec muts obs immediately
but cannot obs effect of LOF mut in essential genes (die)
how to obs effect of LOF mut in essential genes?
conditional mutants where the mut phen is only expressed in certain conditions –> obs phens in continuous range of conditions
example of a conditional allele/mut
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
how to identify secondary muts that modify the mutant phen? what are the types of screens?
modifier screens (mutagenize a mutant)
1) enhancer screens: more severe mut phen
2) suppressor screens: muts that restore WT
experimental evolution
examples of modifier screens
Su(var) and E(var): mutagenize flies with variegated eyes –> see if eyes are more red or more white
what’s experimental evolution
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
what is synthetic lethality
most extreme type of interacting mutation
two muts separately are not lethal, but if both present in one indiv –> lethal
who discovered synthetic lethality? what phenotypes?
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
2 poss explanations of synthetic lethality
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)
how to identify causal mutations via sequencing
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)
2 drawbacks of fwd genetic screens
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)
how can you avoid the two drawbacks of fwd genetic screens
experimental evo
wide range of natural muts
select against muts that decrease fitness
why do reverse genetics?
genomes are redundant so one mutant phen could be from muts in several dif genes
how to make precise genetic edits? why does it work?
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
where did crispr come from
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
how does crispr work in bac and arch
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
can crispr arrays be passed on to offspring
yes
how to apply crispr-cas9 for science
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)
why is Cas9 highly specific? can we still get off target effects?
single mismatch is usually enough to prevent Cas9 from cutting but genomes are redundant so can cut at wrong copy of right sequence
applications of crispr-Cas9
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
implication of RNAi for reverse genetics
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
how to achieve heritable gene knockdown
insert miRNA transgene into org’s genome
what’s a good organism for RNAi? why?
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
what are chimeric genes?
genes in a different context - under a different gene’s regulatory sequences –> ectopic expression
example of chimeric gene? what does the result tell us?
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
Is fwd or rev genetics more amenable to random mutagenesis and crispr-cas9? explain
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.
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.
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
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).
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).
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?
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.
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?
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.
