Techniques of Genomic Manipulations

Question 1

Techniques of genetic manipulation of a random gene

Answer 1

Random mutagenesis screening using chemical mutagens
Random mutagenesis using transposons
Large scale RNAi screening

Question 2

Techniques of genetic manipulation of a specific gene (ad hoc approach)

Answer 2

Exogenous expression of a gene via transgenesis
Targeted disruption of gene via HR (knock out)
Targeted modification of a gene via HR (knock in)
RNAi
Example to figure out what part of the enzyme is the active site

Question 3

What can be modified/altered when interfering with a gene

Answer 3

Modifying expression level of a gene (under/over expression)
Modifying expression pattern of a gene (in time and space)
Modifying protein product of a gene (CDS mutations)

Question 4

What does throughput mean?

Answer 4

How many experiments can physically be conducted
If phenotype is obvious from first look = high throughput screen
Time consuming experiment = low throughput

Question 5

Eyeless gene in drosophila

Answer 5

In drosophila: wild type is red eyes
Mutant is eyeless
Eyeless is responsible for formation of the eye
If eyeless gene is expressed in other parts of the body, an eye will form there

Question 6

How does EMS work?

Answer 6

EMS (Ethyl methanesulfonate) creates mutations in the genome
GC base pair –> EMS adds a group to O of G base –> creates O5-ethylguanine that is recognized as an A base –> Now is an AT base pair
Allows precise random modifications

Question 7

EMS in drosophila

Answer 7

EMS given to male drosophila and modifies all cells (incl. Germline)
Male flies mated to female
Gives rise to mutant progenies
Mutant crossed with a balancer
Mutant mapping and identification of genes

Question 8

Advantages of using EMS

Answer 8

Safe to use
Very efficient
Creates many mutations (on average one mutation every 5-10kb)
Creates random mutations but one mutation may be interesting to study

Question 9

Drosophila life cycle

Answer 9

Drosophila are easy to work with due to quick life cycle
Life cycle lasts 12 days at 25 degrees
Male and female fly mate
Female flies are responsible for choosing their partner
Female fly lays embryos
Takes 24 hours for embryo to give rise to larvae
Larvae stage last for 5-6 days: eats continuously, grows in size
1st, 2nd, 3rd instar larvae
Metamorphosis: once larvae has grown to appropriate size grows pupil case around it
Get a new adult
Takes 24 hours for new adult to be sexually mature
Drosophila is good because have enough time to study larvae and adult flies

Question 10

Genetic screenings in drosophila sorted by throughput

Answer 10

Random mutagenesis with EMS and then screening for phenotype development
Medium throughput = Olfactory (check if flies react to an odor)
Low throughput (time consuming)= Response to pain/body wall muscle (need to teach the fly, wait a few days, see if fly recalls information)
High throughput (efficient): Lethality, body size

Question 11

Transposable elements for mutagenesis

Answer 11

Random mutagenesis
Transposable elements are fragments of DNA that can insert into new chromosomal locations
Often make duplicate copies of themselves in the process
Comprise 50% of human DNA
Responsible for spontaneous mutation, genetic rearrangements, horizontal gene transfer of genetic material
Speciation and genomic change
Cells must repress transposition for genetic stability

Question 12

P-elements in drosophila

Answer 12

Drosophila has a particular type of TES called P-elements
P strains are found in the wild
M strains are found in the lab
Crossing P flies to M flies results in sex selected hybrid dysgenesis
Meaning that the effect of transposons on the genome will be different when crossing male transposon (P) with female (M) or female transposon (P) with male (M)

Question 13

Sex-specific alternative splicing of P-elements in Drosophila

Answer 13

Gene for transposon can also encode for its own repressor via sex-specific alternative splicing
P-element contains 4 exons separated by 3 introns
In germline of female: intron 3 remains –> shorter protein (66kD) which is a repressor –> no transposable elements
In germline of male: complete splicing –> full length protein (87kD) –> transposase –> can jump around in the genome –> introduce random mutations –> sterile progeny

Question 14

P male x P female cross

Answer 14

Repressor from P female prevents transposition of all P-elements

Question 15

P male X M female cross

Answer 15

P element synthesizes transposase and M female does not produce repressor –> sterile progeny

Question 16

M male x P female cross

Answer 16

Repressor from P female prevents transposition of all P elements

Question 17

Transgenesis by transposons in drosophila to make transgenic animal

Answer 17

Use two plasmids to provide control
One is a vector and one is a helper
Vector: Recognition sequence + gene of interest (white+ that gives red eye colour)
Only injecting vector would give no red eyed progeny
Helper: contains transposase which recognises recognition sequence (inverted repeats) and integrates gene of interest into the genome randomly
Cross with white eye host strain
Select transformants
Don’t put transposase and gene in the same plasmid because we want to limit transposase activity

Question 18

Injecting a developing drosophila embryo

Answer 18

Start with single cell fertilized egg (zygote)
First nuclear division (syncytial blastoderm) to create many nuclei in the same cytoplasm/cell
Nuclei migrate to periphery (cellular blastoderm) –> want to inject here to make germline changes
Cell division to create embryo

Question 19

How can the P element be used for Transgenesis in Drosophila?

Answer 19

P-elements insert themselves in Drosophila genome
P-elements can be used to insert a gene of interest
Transposase must be present at moment of injection
But transposase must not be present after injection or for too long
Inject two independent plasmids in the embryo
One encoding for transposase and one with gene of interest flanked by recognition sequences
Gene of interest will insert itself in germline and progeny will inherit it
Stable transgenic is made
Transgenic individuals can be recognised by a collateral dominant marker
Usually mini white gene conferring red eyes

Question 20

What would happen if a transposable element inserts itself in specific regions in the genome?

Answer 20

Intergenic region: Influences expression
Between enhancers: may interfere with ability of TFs to bind enhancers
Before/after 5’UTR: mutant gene/frameshift so protein is not produced. Most common place of P-element insertion
Junction between exons and introns: mutant gene/frameshift so protein is not produced
Within an intron: normally spliced away so doesn’t affect product but can affect splice site

Question 21

Advantage of P-element insertion mutagenesis

Answer 21

Easier to find culprit
Random point mutations everywhere in the genome leads to specific phenotype
Need to identify gene causing the phenotype using gene mapping which takes very long
Using P-elements tells you that gene causing phenotype is the gene that contains the P-element
We know the sequence of the P-element

Question 22

GAL4-UAS system

Answer 22

GAL4 is a transcription factor activator that binds to UAS cis-regulatory sequence
GAL-4 randomly inserted next to a to enhancer using P-elements
GAL4 can insert itself into 5’UTR of a gene so it is then regulated by the enhancer of that gene
GAL4 is now expressed in particular cells/tissues
Example if it is a gene expressed only in macrophages then all macrophages in transgenic animal will express GAL-4
Use P-element to insert UAS coupled to the gene X
So wherever GAL4 is produced (specific tissue) it will bind to UAS leading to expression of gene X
Cross male with GAL4 and female with UAS so part of the progeny will have both and express gene X

Question 23

Transgenic mice

Answer 23

Microinject DNA into the nucleus in the egg as soon as it has fertilised
Implant embryo in foster mother
Embryo gives rise to transgenic progeny
This technique is quite inefficient
Only 5% or less of treated eggs become transgenic progeny
Need to check mouse pups for DNA (by PCR or southern blots), RNA (by northerns or RT-PCR) and protein (by western or by some specific assay)
This takes very long and is very expensive in mice vs in flies experiments are quicker

Question 24

Gene targeting by Homologous recombination

Answer 24

Efficiency of transgenesis is low in every animal
Exploits mechanism of DNA repair that are endogenous to the cell
Works on many organisms from bacteria to mice
Plasmid containing homologous regions that pairs with homologous regions in the genome –> activates homologous repair machinery leading to integration of the gene in the targeted region
Requires access to a stem cell that will become an embryo
Knock out: Can disrupt/destroy the target
Knock in: Can change gene sequence (ex. Changing sequence of changing isoform)
Make it more efficient by adding antibiotic resistance gene
Inject DNA in blastocyst –> creates embryonic stem cells –> add antibiotic to petri dish –> select for cells that have incorporated the gene (successful knock-in or knock-out)

Question 25

Advantages and Disadvantages of using HR

Answer 25

Advantages:
Targeting a specific area of the genome
Can target DNA of any kind
Increases number of embryos that contain the gene of interest
Can be improved by artificially coupling a DNA break to it

Disadvantages:
Success rate still low and still takes very long
Difficult to use for condensed chromatin states

Question 26

Historical principle of RNA interference using anti-sense RNA

Answer 26

RNA transcription and translation are steric activities (involve molecular machinery)
Can interfere with machinery and stop it from working
By providing antisense version of the messenger, messenger will become double stranded so machinery will not recognise it and prevent target from being translated
Ex. antisense RNA to inhibit activity of thymidine kinase

Question 26

DNA plant technology corp.

Answer 26

Manipulating plant genomes for business
Famous controversial products: vine mini peppers, fish tomato, Y1 tobacco (tobacco with stronger addiction properties)
Created a variant of flowers with patterns by interfering with the level of expression of the enzyme that gives flowers colour via antisense RNA

Question 27

Production of antisense RNA in C. elegans

Answer 27

Production of antisense RNA leads to effective inhibition of gene expression in C. elegans
Unc genes are needed for muscle/neuronal function in C. elegans
Unc mutations causes C. elegans to move in an uncoordinated way
Use of antisense plasmid to knock-down unc-22 and unc-54 which led to strong twitchers and slow/paralyzed movement
Injection of sense RNA also induced Unc mutations

Question 28

Genetic interference by double stranded RNA

Answer 28

Injected double stranded, sense, and antisense RNA and only double stranded RNA resulted in a specific phenotype
Mixture of sense and antisense was at least much stronger than the single sense or antisense
dsRNA was shown to retain silencing activity
Injecting two strands with a time interval between led to no silencing (sense and antisense need to be injected together)
Coinfection of dsRNA unrelated to unc-22 did not potentiate the effect of single unc-22 strands so targeting is specific
dsRNA targeting promotor of introns did not lead to silencing (need to target DNA after splicing)
mRNA of targeted genes was greatly reduced in expression so not just reduced translation but also reduced gene expression

Question 29

Genetic approaches to identifying molecules involved in RNAi

Answer 29

Worm ectopically expresses GFP
Add double stranded RNA against GFP
Get knock-down of GFP (now worm is not fluorescent anymore)
Can start conducting random mutagenesis to look for worms that no longer loose fluorescence when given a double stranded RNA molecule
Get genes that are involved in silencing of RNA

Question 30

Biochemical approaches to identifying molecules involved in RNAi

Answer 30

Insert dsRNA into a cell
Pull down one component that is associated with dsRNA
Use mass spec to identify proteins and find binding partners

Question 31

RNAi pathway

Answer 31

RNAi is the use of small RNA molecules to direct gene silencing
Genes are transcribed by RNA polymerase II and splicing produces mRNA
mRNA is translated by ribosome to form proteins
Small interfering RNAs (siRNAs) and microRNAs are derived from longer double stranded RNA and bind to Dicer
Dicer is an endonuclease that cuts RNA into short pieces
siRNAs and microRNAs are approx. 21 nucleotides long
Short dsRNA binds to argonaut protein
Helicase unfolds/unwinds dsRNA
Only one strand remains bound to argonaut (guide strand)
Dicer and argonaut binds with other proteins to form RISC (RNA inducing silencing complex)
siRNAs: produced in the lab, direct RISC to bind to specific mRNAs with full complementarity –> mRNA is cleaved and degraded –> No protein
microRNAs: produced in the nucleus, RNA folds back on itself to form hairpin, Drosha exports from nucleus, DICER recognises hairpin and cleaves it out, direct RISC to bind to target mRNA with only partial complementarity –> allows cleavage of a lot of different mRNAs –> translational repression but not destruction of mRNA –> No protein

Question 32

Dicer

Answer 32

Dicer binds to double stranded RNA
Endonuclease
Cleaves dsRNA into fragments 21 nucleotides long
Specificity to double stranded RNA can come from a viral infection, nucleus or artificially in the lab

Question 33

microRNA during development

Answer 33

partial complementarity between microRNA and target mRNA
so microRNA can target multiple transcripts that encode proteins of the same family
microRNA target genes that have a common 3’UTR
Binding of microRNA and target in seed region of microRNA

Question 34

miRNA and siRNA occurrence in organisms

Answer 34

miRNA occur naturally in plants and animals, is single stranded
siRNAs occur naturally in plans and lower animals but don’t know if they occur in mammals, are double stranded

Question 35

Mechanism of translation inhibition (no degradation of target mRNA)

Answer 35

Interference with initiation complex binding to CAP
Block circularization of mRNA
Decapping and mRNA degradation
Competing for initiation factors
Force ribosome to drop off
Interference with translation: binding of miRNA interferes with ribosome

Question 36

What are the two outcomes of the RNAi pathway?

Answer 36

Cleaving of target mRNA
Inhibition of translation

Question 37

Other types of small RNAs

Answer 37

Many types of small RNAs that have different functions
Example nastiRNA involved in post-transcriptional regulation of genes
Primary and secondary siRNAs (only in C. elegans and plants)

Question 38

Primary and secondary siRNAs

Answer 38

mRNA target binds RNA dependent RNA polymerase which produces fragments of double stranded RNA
When degradation occurs get dsRNA that is recycled to make siRNAs
This is why C. elegans showed strong effects when dsRNA was inserted

Question 39

What are zinc finger nucleases?

Answer 39

Component of many TF as it helps recognise DNA
Zinc finger nuclease is made of zinc finger domains fused with a nuclease
Zinc finger is recognised by a specific region of DNA
Each Zinc finger nuclease interacts with 3 nucleotides
If not all the interactions are created, then cutting is inefficient
FokI only cuts single strands of DNA
If want to make DSB then need to engineer two FokI enzymes to cuts on both strands
Need a DSB to recruit HR machinery and increase efficiency

Question 40

Why are zinc finger nucleases inefficient/difficult to use?

Answer 40

Difficult to engineer two enzymes because don’t have a good understanding of the interaction of zinc finger domains with DNA
Use in silico methods
In vitro evolution (start with sequence that should work and evolution the rest
Example antibiotic resistance is only activated by successful binding)
May have off-target effects if enzyme has poor affinity

Question 41

What are TALENS?

Answer 41

Same goal as zinc fingers but much easier to produce
Consist of a DNA binding domain (TALE) and a nuclease domain
Each DNA binding domain is identical to all others with the exception of two amino acids that have specific binding preference towards a base
Protein is engineered to place domains one after the other and match the code so the domains bind to the nucleotide that you want to target
Each repeat in the DNA binding domain is specific to a particular DNA base
Nuclease domain contains FokI nuclease
Acts as a dimer to create a DSB
Cell’s natural DNA repair mechanisms are activated

Question 42

What are the different components of the CRISPR system?

Answer 42

Cas9 complex made of Cas9 nuclease and single guide RNA (sgRNA)
Cas9 complex binds to protospacer adjacent motif (PAM) sequence
sgRNA unwinds the double helix
RNA strand is designed to match a particular sequence in the DNA
Cas9 cuts the DNA: two nuclease domains create a DSB
Cell repair mechanism often introduces mutations causing the knock out of specific genes

Question 43

Advantages of CRISPR

Answer 43

Quicker and more efficient
Only requires the use of one nuclease
Pairing between nuclease and target DNA does not require a specific protein domain (it is regulated by guide RNA)

Question 44

Guide RNA

Answer 44

Artificial molecule created in the lab mimicking crRNA-tracrRNA pairing as found in bacteria
Guide RNA binds to target sequence
Guide RNA has a constant region (tracrRNA) and a variable crRNA sequence
Can change sequence of target specific crRNA sequence in the gRNA to match the target DNA sequence
Advantage: Creating RNA molecule is straightforward

Question 45

Structure of Cas9 nuclease

Answer 45

RuvC domain and HNH domain sandwich around one strand of DNA each
C-terminal domain binds to guide RNA
Need a GC base pair at the 5’ end of DNA
GC binding is preferred because it is stronger than an AT base pair
Need a PAM sequence at 3’ of DNA
PAM varies for different enzymes
Here it is NCC
PAM is the limiting factor because you need to target a region where the cleavage is 3-4 nucleotides away from PAM sequence

Question 46

Different ways to use Cas9

Answer 46

Can fuse other enzymes onto Cas9 complex to change specific base pairs
Can modify Cas9 to deactivate nuclease domain and instead add transcriptional activator or repressor and increase/decrease transcription of the gene. Can directly activate RNA polymerase or change epigenetics to activate transcription

Question 47

What is a double nick induced DSB?

Answer 47

Alter Cas9 by removing one of the two active sites to create a nickase (makes single stranded breaks)
Double-nick induced DSB: two proximal nicks on opposite strands
Two different gRNAs bind to opposite strands
Need to be in close proximity to bind the same Cas9 nickase
Can be a few base pairs apart
Used to reduce off-target effects

Question 48

How are gRNAs engineered to give different effects of double-nick induced DSB?

Answer 48

If gRNAs are further away it creates mechanical tension so break is not blunt so difficult to repair: HDR will be poor and chance of inducing a mutation is higher. This is useful when creating a knockout
If one gRNA has poor specificity (site C gRNA can also bind to site A): This will only lead to a single stranded nick which the cell can easily repair. Off site targets occur but leads to very little damage
Very rare for two gRNAs to bind and create an off-target effect. But likely to happen in motifs (can target another protein of the same family).

Question 49

DNA repair mechanism

Answer 49

After a DSB occurs, can either undergo Homology directed repair (HDR) or nonhomologous end joining (NHEJ)
NHEJ: cell wants to rejoin the two ends of the DNA which accidentally creates indel mutations (and can lead to frame shift mutations)
HDR: uses a repair template. Example can add a GFP or antibody tag
Use these repair mechanism with ZF, TALENS or CRISPR-Cas9

Question 50

CRISPR as a natural process in bacteria

Answer 50

CRISPR originated from the bacterial adaptive immune system
Phage infects bacterium
Phage has a sequence with a PAM site followed by a protospacer
CRISPR machinery in bacterium cuts PAM sequence
Protospacer is inserted into the CRISPR locus of the bacterium
CRISPR locus contains Cas genes followed by protospacers that the bacterium has cut/recognized before
Now protospacer is called a spacer in the CRISPR locus
CRISPR locus with spacers is transcribed into a pre crRNA transcript and cleaved into fragments of crRNAs
crRNAs bind to Cas9 to guide Cas9 to protospacers it has encountered before
Specificity is loose enough to recognize new phages but doesn’t attack itself

Question 51

How is a gRNA made? What is the genomic CRISPR locus?

Answer 51

Genomic CRISPR locus: TracrRNA, Cas operon (contains coding sequences for Cas family), then CRISPR repeat spacer array (short palindromic repeats intersperced with spacers which are unique DNA sequences derived from phages that the bacteria has encountered in the past)
pre-crRNA: long transcript containing multiple spacers and short palindromic repeats
tracrRNA binds to palindromic repeats between spacers in pre-crRNA
RNaseIII cleaves to create crRNA made of spacer and short palindromic repeat
guide RNA: tracrRNA and crRNA
guide RNA recruits Cas9 to sequence recognized by PAM site
PAM site is important for unwinding DNA so it becomes accessible to rest of guide RNA and Cas9

Question 52

What are protospacers, PAMs and spacers?

Answer 52

Protospacer=fragment present in the phage
PAM=Protospacer Adjacent Motif
Spacer=once PAM is integrated into the genome, there is a bacterial region complementary to the target

Question 53

Different types of CRISPR

Answer 53

Cas9 belongs to type II family
Type II: works in the cell without any accessory component (Requires RnaseIII which is already found in the cell)
Type I: Requires Cas6 and Cas3 family instead of Cas9
Type III: Requires only Cas6