Giorgio Gilestro Flashcards
What does Dr Giorgio Gilestro lecture series focus on?
Gene and Genome Manipulation
What are the two main categories of in vivo genetic manipulation?
What refering to ‘interfering’ with a gene, what are we refering to?
What is the main difference when it comes to wanting to interfer with a specific gene and random genes?
What are the different experimental techniques associated with Interfering with a random gene vs. Interfering with a specific gene?
What are the new frontiers in genetic modification?
- Zinc fingers
- TALENs
- CRISPR - especially relevant
How is a genetic screen performed after intefering with random genes?
What is a modifier screen?
Principles of chemical random mutagenesis - what reagent is most commonly used? How does it result in mutations?
When using EMS to perform random chemical mutagenesis, why do we target germline cells?
Example - Drosophila
Outline the different stages in the Drosophila Life cycle?
Why is Drosophila particular useful organism to study?
During it develop it provides us with 3 Genetic models
- Embryonic
- Larval - simpler Nervous system + easier to study behaviour
- Adult fly
Whenever we run an assay, why do we perform using a high throughput?
Being able to perform reliable experiments repetively and simulatneously - produce a lot of data quickly
What are transposable elements?
How did transposable element lead to the creation of two Drosophila strains that produce sterile offspring?
Why does crossing P Male x M Female lead to sterile offspring? How come M Male x P Female can produce fertile offspring?
Using Drosophila as a model, how are transposon-based modifications/mutations normally performed?
- Transgenesis
What is the difference between Transgenesis and Gene targetting?
Note - both of these involve interfering with a specific gene - ad hoc
We are inserting/deleting a specific gene, e.g. which we might now is associated with the disease, and study it impacts for carefully!
Transgenesis - insertion of a gene into the genome and seeing it impact on the progency - ad hoc
Gene targetting - target specific genes (e.g. HR - Knock in & out) - to study function of gene + allows us to create models of human genetic diseases (see how this specific gene may contribute to the phenotype)
How come when we inject tranposable elements into drosophila embryo - it does not lead to genetic instability (random jumping gene moving about)?
How can the P element be exploited for Transgenesis in Drosophila?
Basically we take advantage of the P element
We introduce it into an embryo using a plasmid (no replication origin) as well as with another plasmid contianing our gene with the recognition sequences
The gene will be inserted randomly in the genome - examine what impact this gene has -
Note - we introduce a collateral dominant marker which allows us to select transgenic animals - basically making sure that the transposon actually inserted
We can cross our transgenic offspring to end up with a stable line off offspring which we can use to study!
Outline what would happen if a transposon inserted itself in one of the regions labelled below?
Explain the Gal-4 UAS system?
Are transposable elements used in Mice?
Transposable element mainly used in Flies not in Mice
Mice - fuse enhancer element with gene - inject into embryo and hope that it inserts by chance (no transposable elements)
Outline how transgenesis is performed in Mice?
What is the efficiency of transgenesis in mammals?
Outline the Gene tragetting (Knock in/out) can be acheived using HR?
System relies on Homologous recombination - flanking region must be surrounded with Homologous DNA to allow for succesful incorporation into the genome
What are the three main intended outcomes for gene targetting via HR?
Summarize Gene Targeting via HR? How is it performed? What animal model is it most commonly used in? What cellular mechanism does it rely on?
Can the RNAi technique be used for unbiased and adhoc genomic manipulations?
BOOOOOYEAHHHHH
When we say that RNA transcription & translation are steric activities, what do we mean?
Was was the first hypothesis of RNA interference using anti-sense RNA put forward?
What were some famous controversial products made by DNA Plant technology corp using RNAi?
What paper did DNA Plant technology corp. publish with regards to RNAi?
What experiment did Fire et al. paper (1991) perform on C. elegans usign RNAi - what was the controversial result?
What did Fire et al. (nobel prize winning paper) reveal about how RNAi works? Why were all previous experiments producing mixed results?
What were the results and conclusions from the Fire et al. 1998 paper that won the nobel prize?
What are the two approaches to identifying molecules involved in RNAi pathway?
What proteins are involved and how can we identify them?
What were to two key proteins identified by Hammond et al. in the RNAi pathway?
What are the two main RNAi pathways?
Briefly outline the siRNA pathway?
RNAi pathway - results from injecting dsDNA!
- Double stranded SiRNA present in cytoplasm
- DICER will cut the siRNA into pieces – same length (21-22 Bases)
- dsRNA is unwound
- Anti-sense Fragment binds to RISC
- RISC binds to mRNA target using the siRNA cleaves it
Outline the difference between miRNA and siRNA?
Explain what is going on in the attached picture - siRNA & miRNA pathway!
Why does miRNA not always show 100% complementairty? Give an example of why this might be useful?
What are the two outcomes of the RNAi pathway?
What are the different mechanism by which miRNA can inhibit translation?
What are some other small RNAs?
How does the RNAi pathway differ between humans and C. Elegans?
Hint - Signal Amplification!
How is RNAi Screening performed in Humans, Drosophila and C. elegans?
Reverse genetic - Linking this back to understanding the function of Genes
We have a specific sequence that targets a known gene but we want to find out what effect it has on the phenotype!
Analyse the Wells for changes in phenotypes
When performing RNAi screening why can’t long dsRNA be injected into Humans like in Drosophila?
What are some example morphological phenotypic effects one can observe using RNAi and mutant drosophila?
What phenotypes are screened using RNAi?
What is the difference between a classic genetic screen and a RNAi screen?
Classic genetic screen - induce random mutation and screen for phenotypes
What is the Drosophila genome composed of?
How does recombination occur in Drosophila?
How is genetic mapping (locating genes) via recombination performed in theory? What are the pracitcal aspects we need to consider?
How is genetics Mapping through Recombination actually done in Drosophila?
Used to map locations of particular mutations
Phenotype? Try to quantify the phenotype rather than qualitatively
E.g. give it a percentage - use computer technology to quantify Eye development - what percentage of eye is missing?
Outline how inverse PCR or Splinkerette can be used to map transponal insertions.
Used to map where transposons have inserted into the genome
Does RNAi require mapping?
What are things you should do when constructing your interfering RNA?
What do the new new frontiers of genetics modification allow us to do?
What is the principle behind using Homologous recombination as a tool for gene targetting?
What do Zinc Fingers & TALENS have in common?
Outline how zinc finger nucleases allow us to create dsDNA breaks? How are they created?
Outline how TALENS allow us to create dsDNA breaks? How are they created?
What are the different parts of the CRISPR Cas 9 complex?
Outline the CRISPR-Cas9 structure.
Basic Idea about how CRISPR Cas9 works? How can this action be modified?
Normally, Cas9 binds to the gRNA –> directs it to a specific DNA sequence (base complementarity) –> Two nucleases in the RuvC and HNH domain can cleave the phosphodiester backbone resulting in a dsDNA break - stimulates repair mechanism of the cell
But! Cas9 can be modified to perform different functions
- Disactivate Nucleases - CRISPR-Cas9 complex targets a specific DNA sequence - add fluorophore to confirm presence of a sequence
- Couple Cas9 protein with specific transcription factors (activators/repressors) - target a particular DNA sequence using gRNA recognition and examine TF imapct
- Couple Cas9 protein with a protein that performs point mutations - allowing for the mutaiton of a specific DNA sequence!
These are just some examples!!!
How can CRISPR Cas9 be modified to increase it’s accuracy for the target sequence?
Outline how creating a Double stranded break using the New Fronteir methods (Talens, Zinc finger & CRISPR) can lead to the introduction of mutations?
Outline what CRISPRs natural role is inside prokaryotic organisms.
What are the different componenets of the CRISPR locus (Cas genes + CRISPR repeats)?
Outline what happens after CRISPR locus activation - how do we end up with an active CRISPR-Cas9 complex?
Define the following words associated with CRISPR-CAS9
- Protospacer
- PAM
- Spacer
- Pre-crRNA
- crRNA
- TracrRNA
- gRNA
Are there any other CRISPR families?
What are the two main things that differ between the different classes of CRISPR?
Who were the peeps involved in the CRISPR drama series?
What was the main thing that won Charpenteir’s & Doudna’s the Nobel prize?
What were the first set of experiments performed by Doudna & Charpentier? - Figure 1
What were the second set of experiments performed by Doudna & Charpentier? - Figure 2
What were the third set of experiments performed by Doudna & Charpentier? - Figure 3
What were the fourth set of experiments performed by Doudna & Charpentier? - Figure 4
What were the fifth set of experiments performed by Doudna & Charpentier? - Figure 5
What did Feng Zhangs paper show? Why does he think it deserves the nobel prize?
In Strategies for gene disruption in Drosophila – Lin et al., 2014 –> How did they divide up the different techniques for genome manipulations?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> How is Random mutagenesis using EMS performed?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> Problems with Random mutagenesis using EMS?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> How is Transposon-mediated Random mutagenesis performed?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> Benefits and problems with Transposon-mediated Random mutagenesis?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> Why is Ad hoc – Targeted Mutagenesis useful and how is it performed in Drosophila?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> How is HR mediated genomic disruption performed in Drosophila?
Fly line containing a donor DNA that comes from a P element Cassette which contains DNA sequences that are homologous to the target flanked by FLP recombinase target sites (FRTs) & I-SceI recognition sites (Plasmid 1)
Two enzymes, the site-specific recombinase FLP and endonuclease I-SceI, are subsequently introduced into the Drosophila line in order to create double stranded breaks in an inserted donor transgene (Plasmid 2)
The recombination between two FRT sites excises the cassette out from the original site, whereas cleavage at the I-SceI recognition site(s) creates a DSB
The double stranded break induces the homology repair machinery to repair the DSB –> results in the homologous recombination between donor DNA and chromosomal sequence – Which can either be End in or End out
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> What determines where knock in or knock down is performed (Ends-in or Ends out)?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> Limitations of Homologous recombination to replace genomic DNA in Drosophila?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> When a dsDNA break is created what are the different outcomes?
- NHEJ –> lead to a indel mutation - frameshift
- Homologous recombination with…
a) endogenous template
b) exogenous template –> allows us to introduce gene modifications in a base pair precise fashion
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> How can Zinc finger Nucleases or Talens be used for targetted mutagenesis?
Strategies for gene disruption in Drosophila – Lin et al., 2014 –> Talen is superior to Zinc finger, But why?
Apart from recombinases (e.g. FLP) what other technique can be adopted in Drosophila for transgenesis?
How can the phiC31 integrase system be used for target gene insertion?
ΦC31 integrase can catalyze the site-specific integration of attB-containing plasmids into so-called attP-containing docking' or landing’ sites located in the genome
How does this work out in Drosophila?
- attP docking site integrated with a transposon into the fly genome
- ΦC31 integrase is expressed given that ther is a mRNA source
- Introduce a plasmid containing our gene of interest with attB site
After this discovery…
- Numerous docking sites were created (fly genomes containing an attP site) –> eg. piggyBac backbone (Venken et al., 2006) & Mariner backbone (Bischof et al., 2007)
- Bischof et al., 2007 - manage to created a fly line with an endogenous source of ΦC31 by using ΦC31 integrase-mediated transgenesis itself into attP docking sites
What do miRNA, siRNA, piRNA (RNAi pathways) all have in common?
Molecular mechanisms of RNA interference – Wilson & Doudna, 2013
Outline how miRNA are processed and delivered to the cytoplasm?
miRNA - Nucleus:
- Pri-miRNA is cropped by the microprocessor complex, comprising Drosha and DiGeorge syndrome critical region gene 8 (DGCR8), a protein containing two double-stranded RNA-binding domains
- DGCR8 recognizes the pri-miRNA’s junction of stem and single-stranded RNA, which likely aids in positioning Drosha for the endnucleolytic cleavage
- Pre-miRNA associates with transport facilitators Exportin-5 and RanGTP and is exported to the cytoplasm
Outline how miRNA and siRNA are processed in the cytoplasm and leads to mRNA silencing?
In the Cytoplasm…
Processing pathways converge for miRNA and siRNA
- Starts by trimming both RNA molecules down to dsRNA that is of the appropriate size to load onto the Argonaute protein - Performed by Dicer enzyme
- Result? 21–25 nt strands, bearing a 2 nt overhang at each 3′-terminus and a phosphate group at each recessed 5′-terminus
Note – In the cleavage reaction & loading onto Argonaute Dicer may be aided by dsRNA-binding protein (dsRBP) – For example, TAR RNA-binding protein (TRBP)
- dsRNA helix is presented to Argonaute, 3′-terminus and 5′-phosphate of the guide strand are bound by the protein’s PAZ and MID domains, respectively, generating the RISC - While the guide strand binds the passenger strand is discarded
- RISC performs surveillance of the cell – binding to ssRNA which is complementary to the Argonaute-bound guide strand - Guide strand nucleotides 2–6 constitute the seed sequence and initialize binding to the target.
The next steps will depend on the degree of complementarity…
Perfect complementarity – cleavage of the target can occur if the Argonaute present bears catalytic activity (Ago2 in Humans)
Non-perfect complementarity - translational repression before or after initiation which may be followed by deadenylation and degradation
Commonly additional cellular machinery is needed - GW182 is a key mediatorin recruiting these additional components & localizing/moving this silencing activity to processing (P) bodies in the cytoplasm
Cool kids facts about the Dicer enzyme?
What does it do?
Generate dsRNAs suitable for loading onto an Argonaute protein - 21-25 nts
How does it perform cuts of suitable length?
The PAZ and RNase III domains act together as a molecular ruler to mete out diced strands of RNA appropriate for a given organism’s silencing machinery.
Paz domain is located 65A from the catalytic site - corresponds to ~25nt
What does the Helicase domain in the N-terminus do?
Function in humans still being defined but in Drosophila it recognize either miRNA or siRNA precursors
Plus the Helicase domain contains binding site for a dsRBP such as TRBP in humans.
How does the protein recognize and bind to RNA?
- RNase III domains –> Provide a flat, positively-charged surface that can accommodate a long RNA helix as well as two active sites containing Mg2+ for phosdiester bond hydrolysis
- Crystal Structure - reveals that Paz domain can recognize 2-nt overhang on the 3′-terminus and a phosphate-bearing 5′-terminus - characteristic of RNA molecules in the RNAi pathway
Cool kids facts about the Argonaute?
What does Argonaute do?
Argonaute + silencing gRNA = effector complex known as the RISC
Argonaute’s key functions are recognition of guide strand termini, target cleavage, or recruitment of other proteins involved in silencing.
What is the role of Human AGO clade (group) of Argonaute proteins?
Mediate cytosolic gene silencing while bound to siRNAs or miRNAs
Humans have four proteins in the AGO clade, dubbed Ago1–4. Of these four, only Ago2 exhibits “slicer” activity: the endonucleolytic cleavage
Note - this protein is not required for silencing - other proteins can recruit binding to other proteins that can repress translation, perform deadenylation & degradation
Structure?
Eukaryotic Argonaute proteins adopt a bilobal architecture, with each lobe containing either the N-terminal + PAZ domains or the MID + PIWI domains
How does Argonaute recognize the guide strand?
3’ end of the RNA molecule
PAZ domain to be responsible for recognizing 2 nt 3′-overhangs –> domain provides a pocket to accommodate 2 nt of a 3′-terminus –> extensive polar interactions with the bound RNA’s buried phosphate group and sugar hydroxyls
5’ end of the RNA molecule
MID domain also bears two invariant lysines that recognize the 5′-terminal phosphate present in silencing RNAs
