M3 L16: Fwd and rev genetic screens Flashcards

Question

how can you avoid the two drawbacks of fwd genetic screens

Answer 1

experimental evo wide range of natural muts select against muts that decrease fitness

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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)

Answer 7

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

Answer 8

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

Answer 9

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

Answer 10

insert miRNA transgene into org's genome

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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.

Answer 15

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

Answer 16

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 17

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.

Answer 18

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.