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Genes and Genomics > Genome Manipulation > Flashcards

Genome Manipulation Flashcards

1
Q

Manipulation In Vivo

A
  1. unbiased approach (interfering with random genes)

2. ad hoc approach with hypothesis (interfering with specific gene)

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2
Q

Interfering with a Gene

A
  • modifying expression levels
  • modifying expression pattern
  • modifying protein product
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3
Q

Random Gene Interference

A
  • random mutagenesis screening with transposons or chemical mutagens
  • large scale RNAi screening
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4
Q

Specific Gene Interference

A
  • exogenous expression with transgenesis

- targeted disruption/modification with HR (knock out or knock in)

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5
Q

Genetic Screening

A
  • random gene targeting
  • screen for modified phenotype with no bias in gene selection
  • eg. screen for genes mutated in eyeless flies to show which are key in eye development
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6
Q

Modifier Screens

A
  • mutate mutant phenotype in intermediate cases to find genes where things get worse or better
  • find important genes in pathway developement
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7
Q

Chemical Random Mutagenesis

A
  • alkylating agents
  • affects single base for single gene mutation
  • alkylation of guanine causes repair machinery to switch pairing to T
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8
Q

Fly Screens

A
  • 3 possible genetic models from same fly

- try to use high throughput or modify low throughput screens

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9
Q

Transposable Elements

A
  • fragments of DNA inserting into new chromosomal locations and making duplicate copies of themselves in the process
  • mobile genetic elements
  • middle repetitive DNA
  • cause spontaneous mutation, rearrangements, horizontal transfer
  • cells must stop this to insure genomic stability
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10
Q

P Element

A
  • wild flies have inactive P element in genome
  • M strains in lab flies
  • P element transposon has colonised all wild flies
  • crossing P to M flies causes sex selected hybrid dysgenesis
  • P x P = repressor is produced stopping transposition
  • P male x M female = transposase causes hybrid dsygenesis
  • P female x M male = repressor stops transposition
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11
Q

Transposon Based Random Mutagenesis

A
  • gene for transposon encodes its own repressor via alternative splicing
  • splicing sex specific
  • 2 proteins encoded by 4 ORFS (3 introns)
  • if intron 3 remains you get a repressor (somatic)
  • if intron is cut the transposon binds recognition sequences of P element and jumps (germline)
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12
Q

Transgenesis

A
  • mode of experimentation involving insertion of foreign gene into genome of organism with germ line transmission of phenotype to progeny
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13
Q

Gene Targeting

A
  • produces strains used to study gene function and to create models for human genetic diseases for which the offending gene is known
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14
Q

Transposon Transgenesis

A
  • use red/white eye flies
  • vector contains gene of interest (white+ restoring red eyes) flanked by P element recognition sequences
  • helper contains transposase element which will randomly integrate white+ into genome
  • inject white eye embryos with vector/helper mix
  • backcross injected adults with white eye host strain
  • select red eye color transformants
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15
Q

Transgenesis with P Elements

A
  • Drosophila
  • P elements insert into genome
  • hijacked to also insert gene of interest
  • transposase must be present at moment of injection
  • vector and helper plasmid injected in embryo
  • gene of interest inserted and inherited to make stable transgenic with dominant marker
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16
Q

Aleatory Transposon Insertion

A
  • random location of insertion
    enhancer: change expression or express marker inserted here
    UTR/exons: disrupt exon/gene
    introns: destabilise mRNA or splicing
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17
Q

GAL4 UAS System

A
  • Gal4 is a TF activating UAS to initate downstream transcription
  • specific enhancer controls Gal4 expression
  • cross gal4 and UAS lines
  • UAS gene x can be selected by researchers
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18
Q

Transgenic Mice

A
  • DNA microinjection of transgene into embryo
  • low integration rate
  • check offspring for DNA using assays
  • expression varies based on location/copy number
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19
Q

Gene Targeting via HR

A
  • exploits DNA repair mechanisms endogenous to cell
  • needs stem cell
  • creates knock outs and knock ins
  • coupled to artificial DNA break
  • plasmid contains gene of interest flanked by matching sequences to gene to be disrupted
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20
Q

RNAi

A
  • biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression
  • transcription and translation are steric: disrupting this will stop the process
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21
Q

Fire et al

A
  • found that dsRNA led to phenotype in all progeny
  • ds has silencing activity
  • must be complementary
  • effect is gene specific
  • introns/promoter targets were not affected
  • reduced mRNA levels: transcription and translation was affected
  • genetic or biochemical identification
22
Q

siRNA pathway

A
  • dicer enzyme (RNA nuclease) cuts dsRNA into siRNA (21-22 bp)
  • unwinding : only guide strand retained in cell
  • fragments form RISC complex that interacts with and cleaves target mRNA
  • The siRNA is an exogenous double-stranded RNA uptaken by cells
  • can be endogenous
23
Q

miRNA pathway

A
  • miRNA genes transcribed into pri-miRNA
  • drosha cleaves into hairpin pre-miRNA
  • exportin out of nucleus
  • dicer cleaves miRNA
  • unwinding of dsRNA
  • RISC complex formation
  • translationap repression
24
Q

lin-4

A
  • dicer cleavage of pre-miRNA
  • interference of different points on lin-14 mRNA
  • lin-4 is found on chromosome II in C. elegans and is complementary to sequences in the 3’ untranslated region (UTR) of lin-14 mRNA, this complementary transcript containing seven binding sites
  • lin-4:lin-14 duplex is seen to take up an unusual kinked structure, caused by induced changes in the groove dimension and base stacking due to the mismatched base pairs
25
Q

miRNA

A
  • single stranded
  • not exact complementarity (broad targets)
  • regulate genes other than those that express them
  • inhibit translation
26
Q

siRNA

A
  • double stranded
  • exact complementarity (specific knock-down)
  • regulate same genes expressing them
  • cleave mRNA
  • gene silencing
27
Q

Mechanism of Translation Inhibition

A
  • interference with initiation complex binding to CAP
  • interference with ribosome retention
  • block circularization
  • decapping and mRNA degradation
28
Q

RNAi screenings

A
  • going from gene to phenotypes
  • insert long dsRNA into cells to knock out genes
  • use marker to see chromosomal DNA to look at synthesis and replication
  • use GAL4 system with gene x being an artificial miRNA
    (validate with visible morphological phenotypes)
29
Q

RNAi in Flies

A
  • transformer 2 RNAi and mutant females resemble males anatomically
  • eye absent RNAi mutant males have no eyes
  • stubble males have short stubbly bristles
30
Q

RNAi screen benefits

A
  • no need for cloning or mapping
  • gene sequence known
  • reduced wild type product level
  • not every gene susceptible
  • can insert at different life stages
  • non heritable
31
Q

Food Preference Screen

A
  • RNAi to knock down genes randomly
  • colored protein vs sugar rich foods
  • can see based on color which genes are key in food preference
32
Q

C. Elegans RNAi

A
  • transform gene of interest into vector
    1. transform into E coli and feed worms on individual strains expressing different dsRNAs
    2. transcribe vector and inject dsRNA into gonad
    3. soak worms in dsRNA solution
33
Q

Basic Drosophila Genetics

A
  • 4 chromosomes

- recombination during meiosis

34
Q

Recombination

A
  • parental chromosomes cross over to form new chromosomes

- only in female gametes

35
Q

Balancer Chromosome

A
  • Frequency of recombination proportional to chromosomal distance
  • Difficult to keep stock of two mutations in the lab (except if carried in male lines)
  • Can create homozygous stock (mutations present on both chromosomes so the same product obtained even if recombination occurs (not possible with lethal homozygous mutations)
  • Balancer is an alternative to this
    o Chromosome hit with x-rays to create inversions (piece of chromosome touches itself and inserts in reverse direction)
    o Prevents proper annealing between chromosomes
    o For meiosis HR is needed: screws up other chromosome to prevent meiosis
    o This then keeps both mutations on one chromosome for experimental purposes
    o Can ensure a pure genotype of mutant over balancer – heterozygous lethal mutant females
36
Q

Purpose of RNAi

A
    • Must look at evolutionary purpose: fight dsRNA virus
      o Cell fights dsRNA : something it doesn’t have
      o Cell already has ssRNA in the system
      o Having dsRNA presence triggers the system : defense mechanism created as a response
37
Q

Recombination Mapping

A
  • one WT and one mutant chromosome
  • analyse points of breakage using SNP mapping
  • sequence both to determine bases and pick number of differing bases along length
  • sequence PCR fragments after recombination to examine map of recombination based of positions of WT/M SNPs
  • can link genotype to phenotype
38
Q

Mapping Transposomal Insertion

A

Inverse PCR

  • don’t know insertion location
  • find restriction site in transposon genome (cut transposon in half)
  • ligate fragments that act as primers for PCR
  • from flanking regions you known insertion point of transposon

Splinkerette PCR

  • splinkerette = DNA structure replacing plasmid
  • ligate to restriction ends
  • transposon not cut in half
  • sequence fragment
39
Q

Gene Targeting by HR

A
  • hijack repair machinery to change gene by providing a template for new genomic content
  • need something to repair: artificially damage point of target
  • insert selection marker
40
Q

Zinc Fingers

A
  • genome editing with engineered nucleases (deletion or insertion)
  • induce targeted break by creating nucleases acting on specific sequences
  • unique recognition site
  • binding proteins + FokI nuclease (fusion protein)
  • zinc fingers must all bind for activity
  • nuclease creates sticky end endonuclease
  • finger penetrates pocket of open DNA and binds specifically
41
Q

TALENS

A
  • simpler to make: don’t invent anything
  • subunits bind without interference to other units
  • transcription activator-like effector nucleases
  • nuclease fused to DNA binding domain easily engineered to target any sequence
  • use TALEN end to pair nucleotide sequence with protein modular sequence in which you insert domains
  • FokI creates ss breaks only: need two
  • limitation is that you must clone lots of things together
42
Q

Zinc Finger Modification

A
  • start with finger with similar recognition site
  • produce in bacteria to examine binding
  • in vitro evolution by applying selective pressure until the fingers bind your specific region and not anything else
43
Q

CRISPR

A
  • Cas 9 nuclease: recognises target via guide RNA rather than protein DNA interaction
  • 2 nuclease domains
  • RuvC/HNH domain: guides binding of guide to target DNA/unwinding
  • C-terminal domain: guides binding to guide RNA
  • PAM sequence in target allows pairing
  • 5’ pair between target and guide must be G-C
44
Q

CRISPR Process

A
  1. complex binds DNA
  2. guide unwinds target DNA
  3. match leads to ds nick at 2 active sites
  4. repair machinery of cell is error prone so can cause knock out mutations
45
Q

CRISPR Uses

A
  • knockout mutations
  • transport new enzymes to sequence
  • fused deaminase to target disease gene and introduce a stop codon
  • promote transcription by adding activators (or silencers)
  • attach fluoresence to visualise gene position or structure
46
Q

CRISPR Specificty

A
  • change Cas9 to a ssDNA nuclease to give more specificity
  • use two guides to cause dsbreak
  • lowers off target breaks
47
Q

Non Homologous End Joining

A
  • inefficient and makes mistakes
  • frameshifts
  • mutants created
  • removal or insertion of bases
48
Q

Homology directed Repair

A
  • gives repair template to incorporate new sequence
49
Q

Endogenous CRISPR

A
  • bacterial protection from phages
  • protection from foreign DNA
    1. phase DNA insertion
    2. crispr locus activated (cas genes and CRISPR loci)
    3. transcription of both
    4. RNA fragmented and associated with cas protein
    5. complex activates crispr-cas9 activity
    6. crRNA fragments are guides targeting invasive genome for destruction
    7. invading genome fragments collected and used to incorporate as memory into CRISPR locus (remaining defense)
  • allows adaptive immune response
  • First a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus. The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid.[2] There are several CRISPR system subtypes.
50
Q

Components of CRISPR

A
  1. protospacer: target of foreign DNA
  2. PAM: protospacer adjacent motif
  3. spacer: bacterial region complementary to target (area of guide with specificity)
  4. pre-crRNA: long transcript containing multiple spacers and short palindromic repeats
  5. crRNA: mature pre-crRNA made of spacer and short palindromic repeat
  6. tracrRNA: RNA molecule for pre-crRNA maturation (cuts spacers to connects them to Cas9)
  7. gRNA: artificial molecule mimicking crRNA:tracrNA pairing
51
Q

CRISPR locus

A
  • protospacers interspersed with short palindromic repeats
  • transcribed into pre-crRNA
  • fragmented into crRNA:tracrRNA:Cas9 complexes
  • binds to PAM on target sequence for cut
  • guide RNA made of 2 molecules in wild: tracr/crRNA
  • tracr induces cleavage of precursor by Cas9
52
Q

Type 2 CRISPR

A
  • Type II CRISPR-Cas systems require a tracrRNA which plays a role in the maturation of crRNA.[3] The tracrRNA is partially complementary to and base pairs with a pre-crRNA forming an RNA duplex. This is cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid.

Genes and Genomics (13 decks)

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