Reverse genetics 2 Flashcards
RNA interference
Gene silencing process that is sequence specific and mediated by double-stranded RNA molecules (siRNAs and shRNA/microRNA).
RNAi silencing is at the ______ level
Posttranscriptional
- mRNA degradation and translation repression
When was RNA interference initially observed?
In petunias where overexpression of a pigmentation gene resulted in its own down regulation (co-suppression of gene expression)
The molecular basis for RNA interference was discovered by…
Craig Mellow and Andrew Fire in C. elegans who determined that it was double-stranded RNA and not sense/antisense RNA that cause gene silencing
True or false: RNA interference is ubiquitous in almost all eukaryotes examined so far
True
- It is thought that this molecular mechanism evolved to control spread of viruses and transposons.
Small interfering RNAs (siRNAs)
Long double-stranded RNAs (400-700 bp) are cleaved to 21-23 bp by the Dicer Rnase III (endoribonuclease that only cuts dsRNA) to generate siRNAs.
- siRNAs are unwound and one of the strands functions as a template in RISC (RNA indicuing-silending complex) to guide cleavage of the omplementary mRNA
MicroRNA
Noncoding RNA located in intergenic regions or within genes are transcribed by RNA polymerase II
- Single-stranded RNA is cleaved in the nucleus by Drosha generating a short hairpin mRNA which is then processed by DICER and the RISC complex similar to siRNA
True or false: microRNA is not a major regulator of gene expression, and is not implicated in numerous cellular processes/diseases
False
- MicroRNA was discovered recently to be major regulators of gene expression (>50% of all genes)
- If you delete microRNA genes, leads to misregulation of genes which can lead to diseases.
- Implicated in numerous cellular processes and diseases including differentiation, proliferation, apoptosis, cancer, heart disease.
What is introduced into cells/organisms for systematic knockdown of genes
Introduction of synthetically-designed siRNA/shRNA/microRNA
- Chemically synthesize various RNA molecules for gene silencing
True or false: targeted gene knockdown in multicellular organisms can be adapted for large-scale reverse genetics
True
- complex eukaryotes
True or false: RNAi alleles are knockout/loss of function
False
- RNAi alleles are knockdowns/partial loss of function (hypomorph alleles, not null alleles)
True or false: Every gene is susceptible to RNAi, like deletion mutagens are sensitive to deletions
False
- Unlike other mutagens, not every gene is susceptible to RNAi (Resistant tissues and genes encoding proteins with long half lives)
Multiple genes with shared sequences can be knocked down.
What is an advantage or disadvantage of this?
Advantage: Uncovers redundancy
Disadvantage: off-target knockdowns make phenotypes difficult to interpret
If a gene is knocked out but has a compensatory gene (functional redundancy), will a phenotype be observed?
A phenotype will not be observed. To deal with functional redundancy, you would need to design a different RNAi for the compensatory gene.
True or false: Knockdowns with siRNA are usually heritable
False
- Knockdown with siRNA is usually not heritable and transient unless the transgene producing the siRNA is integrated in the gene.
What does introducing siRNA at different developmental stages allow for?
Makes it easier to study maternal genes with essential zygotic functions (knockdown maternal genes and determine the function of these in development of the fetus; can’t do a deletion because that would be lethal to the fetus so can’t be studied)
How is siRNA introduced into worms?
By microinjection or feeding bacteria expressing the siRNA
Describe how bacteria expressing the siRNA is produced and inserted into C. elegans (5 steps)
- Amplify >19,000 fragments from genomic DNA using PCR
- Insert siRNA gene into T7/fire vector (produces dsRNA, promoter on one side makes sense strand and promoter on other sides makes antisense strand)
- Anneal sense and antisense strand the make dsRNA (anneals because they’re complementary)
- Clone vectors into E.coli
- Put bacteria on plate for worms to eat
Describe the full genome RNAi profiling of early embryogenesis in C. elegans (4)
Identified 661 genes involved in early embryogenesis
- 20, 326 dsRNA were designed and used to target 19075 (98%) of genes
- Screen was performed to identify all genes for mitotic cell division in a metazoan
- Phenotypic analysis was performed by 40,000 differential interference contrast microscopy (movies) about 20 minutes after fertilization until the four cell stage.
Why does RNAi identify a smaller number of essential genes compared to yeast mutant deletion strain experiments?
Knockdowns result in cell viability unlike knockouts done in essential genes.
- Essential genes are still there in RNAi, just not being detected because there’s still some expression of the essential genes in knockdowns (so can’t determine which genes are essential because knockdown of some essential genes doesn’t lead to cell death)
Eliminating any proteins that compromises the function of the same protein complex should result in…
The same consequence or phenotype (guilty by association)
In the full genome RNAi profiling of early embryogenesis in C.elegans, what were 79% of all known ribosomal genes classified as? Explain why
79% of all known ribosomal genes were classified as “severe pleitotrophic defect”
- Means that phenotype is mixed (not very uniform
- Knocking down protein synthesis affects many processes
Genes with common mutant phenotypes in the RNAi profiling experiment are associated with…
Similar chemical pathways and key events of the first cell divisions
What is the goal regarding embryogenesis genes with common mutant phenotypes?
The goal is to further characterize these genes to see how they interact with other genes/proteins and form molecular networks governing embryogenesis.
What do terminal phenotypes tell you in gene silencing for cell division genes?
Allows you to determine where in development the gene actually functions based on the terminal phenotype
True or false: Large scale RNAi screening is practical for mammalian animal models (e.g. mouse, chimps)
False
- RNAi screening is not practical for these types of animal models
Since large scale RNAi screening is not practical for mammalian animal models, what do we have to use instead for full genome RNAi screening?
Cell lines (e.g. knocking down all the cells in the cancer cell line, or the kidney cell line, etc.)
Describe how to get temporary vs stable effect of perturbation in RNAi screening in mammalian cell lines
Temporary (transient):
Transfecting siRNA directly into target cell.
- plasmid with shRNA or shRNA-mir gene can also be transfected into target cell
Not transient:
shRNA or shRNA-mir (microRNA structure in shRNA) get cloned into plasmid, which infects the target cell and inserts DNA into host genome from virus.
What are the two formats for high-throughput mammalian RNAi screens?
- Multi-well-plate-based RNAi
- RNAi-based cell microarrays
Describe multi-well-plate-based RNAi (5 steps)
- Library prepared of bacterial glycerol stock or siRNAs (1 specific siRNA in each well targetting a specific gene)
- Preparation of transfection-quality DNA or siRNAs
- Re-array siRNAs or plasmid shRNAs into 384 - well plates for high-throughput screening (transient) OR prepare shRNA-expressing virus and then re-array virus into 384-well plates for high throughput screening
- Transfection (or reverse transfection) of siRNAs or plasmid shRNAs into target cell lines (transient) or infect shRNA-expressing virus into target cell lines
- Assay phenotype of interest
- Now you know exactly which knockdown contributes to the phenotype (b/c this is reverse genetics)
Describe RNAi-based cell microarrays
- Library of siRNAs, plasmid shRNAs, esiRNAs, and virus shRNAs mixed with printing buffer
- Library is arrayed onto glass slide with microarraying robot
- Printed microarrays of siRNA/esiRNA microarray (reverse transfection), plasmid shRNA microarray (reverse transfection) and viral shRNA microarray (reverse infection) can be stored or used directly (instead of probes, it will be siRNA)
- Microarrays can be incubated with cells and then imaged live, or fixed and stained, imaged and analysed.
On an RNAi-based cell microarray, if there is no cell growth in one region of the microarray, what does this indicate?
The siRNA/shRNA viruses in this region knockdown essential genes
What are two ways of visualizing RNAi screens?
- Reporter assays (Can see the phenotype in response to knockdowns (e.g. luciferase gene, protein modification, protein interaction, morphology, etc)
- Image-based screening
What is the issue with interpreting the results (reporter assays/image-based screening) phenotypic assays for RNAi screens in mammalian cells?
Looking at thousands of cells -> big biology
- can’t do this manually, it’s very tedious
What is needed in order to interpret RNAi screen results?
High-throughput microscopy
- Automated microscope for acquisition of thousands of cell images (so you don’t have to manually take pictures of the wells)
- Automated image analysis with software to find cells and carry out assays including cell size measurements, morphology recognition and fluorescence intensity of biomarkers
- Require specialized hardware and software for handling terabytes of data
Describe how a lentiviral RNAi library of human genes was applied to high-throughput screening (5)
- Lentiviral shRNA library targetting 12,000 human genes, with 5 shRNA constructs/gene (5 shRNA targets each gene)
- Lentiviral vectors are better than other viral vectors since they work in a wide variety of cell types including cancer cell lines
- Evaluated the effectiveness of the shRNA library by testing a subset (4903 shRNA/1028 genes) in array format (one shRNA/well)
- Automated fluorescence microscopy to screen cells with cell cycle defects (histone H3 phosphorylation as an indicator of cells arrested in mitosis)
- This screen identified several known and ~100 candidate regulators of mitotic progression/proliferation
In the lentiviral RNAi library of human genes applied to high-throughput screening, what did the knockdown of CDC2 (one of the major CDKs involved with entry into mitosis) with shCDC2-820 result in?
Arrested cells in mitosis
- Knockdown causes increases mitotic index (% of cells in mitosis) and phosphorylated histone H3, replicated DNA and absence of cdc2 protein
- More cells with 4N content than 2N -> indicates that cells are duplicated and arrested in mitosis -> shows that the method of shRNA is working
What did the lentiviral RNAi library of human genes applied to high-throughput screening help discover?
Potential novel positive regulators of mitosis
- Found by identifying gene knockdowns that caused a delay in mitosis
- All of these genes are potential cancer cell targets -> these genes being knocked down/out would result in decreased cell division
What does imaging the nucleus during mitosis gene knockdowns allow for?
Allows for assigning (based on phenotype) which cell stage each gene functions in.
- e.g. if you knockdown gene A, and the nucleus has late prophase morphology, you can infer that gene A is involved in progressing late prophase
True or false: in multicellular organisms, cell proliferation must be coordinated and occur at different rates for different cell types
True
- The mammalian cell cycle is high regulated (cells only proliferate to a certain extent at certain times).
What does cell proliferation depend on?
Growth factors, rather than nutrients
Unlike yeast, mammalian cells possess multiple ______ called ____ which associate with different ______
Describe the functions of these proteins
Unlike yeast, mammalian cells possess multiple cdc2-like kinases called cyclin-dependent kinases (Cdk1-7) which associate with different cyclins (Cyclin A-H)
- CDKs and cyclins are the proteins that push the cell cycle to progress (there are different CDKs and cyclins in different stages of the cell cycle)
In addition to cyclins, cdks also confer…
Regulation and substrate specificity (each CDK has its own substrate)
True or false: most of our cells are actively dividing
False
- Most of our cells are non-dividing
What happens (in general) when cell cycle regulation goes wrong?
Cancer
Define cancer
Any malignant growth caused by abnormal and uncontrolled cell division, and is caused by altered expression of multiple genes as a result of mutations
What type of disease is cancer?
Polygenic disease
- caused by multiple mutations
Which 2 mutated genes are implicated in cancer? * on final
- Oncogenes
- Tumour suppressors
Oncogenes and 2 examples
What type of mutations of oncogenes cause cancer?
Positive regulators of the cell cycle (gain-of-function) including cyclin D/E (gene aplification), cdk4 (insensitive to CKI inhibition)
- Gain-of-function mutations
Tumour supressor genes and 3 examples
What type of mutations of tumour suppressors cause cancer?
Negative regulators of the cell cycle (loss of function) including checkpoint genes p53 and CHK2, p16^INK4A (CKI) and RB (Whi5 homolog)
- Loss-of-function mutations
About 50% of tumours have what type of mutations?
Inactivated p53 gene and cyclin D and E are often highly expressed in breast cancer carcinomas
How is cancer potentially inherited?
Maybe you’re already born with half of the mutations and gain the other mutations throughout life
How would you identify essential genes in cancer cells?
* on final
shRNA screen for loss-of-function mutations that stop cancer cells from dividing or kill them
Essential genes for cancer cells are… (2)
* on final
- Potential drug targets
- Common in all cancers, as well as cancer specific
What could the identity of essential genes in cancer cells provide?
* on final
Clues to their function and the molecular basis (growth regulatory pathways) behind specific cancers
Describe the pooled-based dropout shRNA screen with barcode microarrays IN GENERAL
- Infect cancer cells with a pooled shRNA library and use a barcode microarray to see which shRNAs cause cancer cells to stop dividing
- Molecular barcodes are gene/probe specific sequence in the shRNA stem (half-hairpin (HH) barcodes)
Describe the DETAILED steps of the pooled-based dropout shRNA screen with barcode microarrays
- shRNA oligonucleotides printed on microarray
- PCR-cloning of cDNA of shRNA into plasmid library
- Packaging plasmids into retrovirus pool
- Infect cells with viruses. Extract all the DNA/PCR barcodes from initial genes present with Cy5 dye, and label genes present after time with Cy3
- Mix PCRed Cy3 and Cy5-labelled barcodes and competitive hybridization to microarray
What is the barcode in the pooled-based dropout shRNA screen with barcode microarrays? What part of the shRNA undergoes PCR and why?
The barcode is the shRNA cDNA (because each shRNA is unique)
- PCR only antisense/primer. Can’t PCR entire hairpin b/c structure will fold back on itself (and won’t hybridize onto probes on microarray)
On a pooled-based dropout shRNA screen with barcode microarrays, what would a dropout on the microarray represent (red spot)?
The gene that got knocked down is likely a positive regulator (oncogene)
On a pooled-based dropout shRNA screen with barcode microarrays, what would an enrichment on the microarray represent (green spot)?
Cancer cell is more aggressive (what we don’t want), so the gene that got knocked down is likely a negative regulator (tumour suppressor)
A screen was performed in normal human mammary epithelial cells (HMECs), two colon cancer cells and a breast cancer cell line. HMEC had the most essential genes than the cancer cell lines. Why?
Cancer cells are more driven to divide so mutations will not impact cell division as much.
- Cancer cells have accumulated mutations that made them driven to divide and they need much more mutations to decrease cell division
A screen was performed in normal human mammary epithelial cells (HMECs), two colon cancer cells and a breast cancer cell line. There were 19 common genes among these cell lines. Would these be good drug targets? Why or why not?
These would not be good drug targets because they’re essential genes in normal cells too, so targeting these genes would kill normal cells too, rather than just cancer cells.
A screen was performed in normal human mammary epithelial cells (HMECs), two colon cancer cells and a breast cancer cell line. There were 19 common genes among these cell lines. What were these 19 genes involved in?
Core cellular processes essential for all four cells (e.g. cell cycle regulation, translation, protein degradation)
A screen was performed in normal human mammary epithelial cells (HMECs), two colon cancer cells and a breast cancer cell line. There were 19 common genes among these cell lines. What types of shRNAs were recovered?
shRNAs against multiple subunits of essential protein complexes were recovered.
Describe CRISPR-Cas9 in general and what it allows for
Dual (2 types of) RNA molecules that guides DNA endonuclease (Cas9) to direct DNA cleavage at a specific site in the genome.
- New method for genetic engineering in unicellular and multicellular eukaryotes
- Allows for precise insertion, deletion and tagging of genes using engineered nuclease/molecular scissors (genome editing)
Genetic manipulation in CRISPR-Cas9 is (transient/permanent) and (heritable/not heritable)
Permanent and heritable
True or false: genetic manipulation in CRISPR-Cas9 involves a transgene, like an antibiotic resistance marker, that remains in the mutant
False
- No transgene remaining in mutant
CRISPR-Cas9 does not allow for a transgene to remain in the mutant. What is this good for?
Good for mutations in food for example, because some people are sensitive to GMOs.
What two things did the bacterial adaptive immune system co-discovered by Jennifer Doudna and Emmanuelle Charpentier contain?
- CRISPR (clustered regularly interspaced palindromic repeats)
- Cas9 (CRISPR-associated) endonuclease
Describe the bacterial adaptive immune system consisting of CRISPr and Cas9 (2 steps)
- Spacers/protospacers originate from plasmid and viral sequences that once infected bacteria
- The spacers are transcribed to form crRNA which bind to Cas endonucleases and direct them to cleave foreign DNA sequences by complementary base pairing
- allows for immunization of eubacteria and archaea, as it allows for memory of all the lifetime infections the bacteria has received.
Describe the mechanism of CRISPR-Cas9 mediated genome targeting
* on final (5 steps)
- crRNA contains a repeat portion and an invader targeting portion (protospacer)
- Trans-activating crRNA (tracrRNA) is required for crRNA maturation by forming a tracrRNA:crRNA duplex that is recognized by Cas9
- RNA-guided Cas9 is directed to target DNA by complementary base pairing of a 20 nucleotide sequence in mature crRNA
- Both DNA strands of target are cut by the Cas9 endonuclease
- Can induce a double-stranded break in a specific region of the genome by using a specific 20 bp crRNA targeting sequence (this can be done to introduce mutations)
What is the “dual RNA” of CRISPR-Cas9?
crRNA and tracrRNA
What part of the crRNA binds to the tracrRNA? Which part binds to the DNA?
CRISPR repeats bind to the tracrRNA and the protospacers bind to the DNA
DNA targeting of the CRISPR-Cas9 system requires a ___ sequence in the actual DNA being targetted
Explain why this sequence is required
PAM (Protospacer Adjacent Motif: 5’-NGG-3’)
- PAM sequence is required for initial binding of DNA target to Cas9
What makes up the guide RNA in CRISPR- Cas9? What two critical features are in the sgRNA?
Combination of dual tracrRNA:crRNA to single guide RNA (sgRNA) that retain two critical features: 20 nt sequence at 5’ end and double stranded RNA at 3’ end that is recognized by Cas9
What can be used to program CRISPR-Cas9 to target any DNA sequence of interest (DSB)? What must hold true in order for this targeting to occur?
20 nt of the sgRNA can be used to program CRISPR-Cas9 to target any DNA sequence of interest (DSB), as long as it is adjacent to a PAM.
What is present in the dual tracrRNA:crRNA to make the joining process of these RNAs faster?
A linker loop between the crRNA and the mature tracrRNA
What are the two pathways by which a double stranded break will be repaired?
- Non-homologous end joining (NHEJ)
- Homologous recombination
Explain non-homologous end joining (NHEJ) in general
Error prone and introduces deletion and insertion (indel mutation)
Explain homologous recombination
Uses the wild-type copy of the chromosome or donor DNA for gene correction
- Homologous recombination: no mistakes made, perfect exchange of genetic material
How can you modulate gene activity using a defective Cas9 and what 3 ways can this be done?
*on final
Can modulate gene activity using a defective Cas9 tethered to a regulator or GFP
1. Genome engineering with Cas9 nuclease
2. Genome engineering by double nicking with paired Cas9 nickases
3. Localization with defective Cas9 nuclease
Describe genome engineering with Cas9 nuclease to modulate gene activity (2)
- Can be used to do non-homologous end joining (NHEJ) for insertion/deletion mutations
- Can be used to introduce any gene you want (for example, wild-type gene) by homology directed repair (HDR). Donor DNA can be expressed from a plasmid for double recombination as well.
Describe genome engineering by double nicking with paired Cas9 nickases
Mutated Cas9 makes staggered single stranded cuts
- Homology directed repair (HDR) -> happens at a much faster rate than normal genome engineering with Cas9 nuclease because recombination machinery is much more effective when the cuts are staggered.
Describe localizataion with defective Cas9 nuclease
Proteins are tethered to Cas9
- Can engineer Cas9 using CRISPR to activate/suppress a specific gene, or can tether GFP to Cas9 to visualize the gene.
True or false: CRISPR (non-GMO) foods are being produced
True
Describe an application of CRISPR to human therapy of sickle cell anemia (4)
- Sickle cell anemia and beta thalassemia is caused by non-functional β hemoglobin gene.
- BCL11A gene is a transcriptional repressor of fetal γ in place of β hemoglobin
- CTX001 inactivates BCL11A by CRISPR/Cas9 in extracted blood stem cells allowing production of γ hemoglobin in place of β hemoglobin. γ hemoglobin can still bind with α hemoglobin to make functional hemoglobin.
- Infuse CRISPR-edited cells into patient
Why is a cure for gene-related diseases bad for businesses?
Only get each patient once -> not as much money being made
What are 6 limitations of CRISPR-Cas9?
- Off-target mutations (other double-stranded breaks can occur elsewhere in the genome)
- Higher rate of NHEJ is okay for gene inactivation, but not for gene correction
- Certain CRISPR-Cas9 procedures can potentially induce oncogenesis
- Efficiency of genome editing is still quite low depending on the type of editing
- Mosiacism in gene-corrected embryos would be difficult to detect and pose safety issues for clinical applications (within the embryo, some cells will have the edited gene and some will not if CRISPR occurs later in development)
- Larger-scale CRISPR-Cas9 screens are feasible, but not routine
