Huang Lectures Flashcards
Gene Manipulation with Mutagenesis
- the process begins with P0 generation exposed to a mutagen, creating genetic variations.
- these modifications are passed down to F1 and F2 generations.
- common wild-type phenotype and rare mutant phenotype differences allow researches to identify genes responsible for specific traits.
What is ‘functional genomics’? and what other fields does it integrate?
- functional genomics uses genome sequence data to understand gene functions and how they interact within biological systems.
- other fields integrated: genomics, molecular biology, bioinformatics.
Forward Genetics
- starts with natural variation or induced mutagenesis
- moves from phenotype (observable trait) to genotype (underlying genetic cause) to pinpoint genes associated with specific phenotypes.
Reverse Genetics
- involves targeted gene modification
- starts with known genotype and investigates the resulting phenotype to understand the gene’s function.
Gain of Function Genetic Screens
- activation/enhancement of gene function
- DNA transfection
- cDNA/ORF
- Insertional mutagenesis
- CRISPRa
- gain of function screens aim to identify genes that, when over expressed or mutated lead to specific phenotypes such as uncontrolled cell growth.
Loss of Function Genetic Screens
- inactivation/suppression of gene function
- classical gene knockout
- insertional mutagenesis
- RNAi
- CRISPR knockout
- CRISPRi
- Cas13d, Cas 7-11 - mediated gene supression
Altered function - genetic screens
- other CRISPR editing tools - DSB mediated, base editing, prime editing.
Example of DNA Transfection (GoF Genetic Screen)
- Human HRAS oncogene
- normally, Ras protein switches between inactive (Ras-GDP) and active (Ras-GTP) states, regulating cell growth and migration.
- mutation-induced gain of function: an increase in the active form (Ras-GTP) can disrupt balance, leading to uncontrolled proliferation and cancer.
Pros & Cons of
cDNA over-expression (GoF Genetic Screen)
1) Pros:
- effective, adaptable, and enables systematic over-expression.
2) Cons:
- can produce experimental artifacts like cloning errors (e.g., gene truncations or mutations) and can cause abnormal expression levels due to the overuse of strong promoters or genes not typically expressed in certain cell types.
What are two types of Insertional Mutagenesis?
- Virus-Mediated:
- Viral vectors (e.g., lentiviruses) insert genes with a strong promoter (viral LTR) into the host genome, causing random mutations.
- These insertions may activate or inactivate genes by adding enhancers, promoters, or disrupting gene coding regions.
- Transposon-Mediated:
- DNA transposons like PiggyBac and Sleeping Beauty can jump into new DNA locations, driven by transposase enzymes.
- This approach enables reversible integration, where transposable elements can deliver genetic elements like promoters, splicing sequences, or transcriptional stops
What is Lentiviral Transduction?
Lentiviral vectors are often used to deliver and integrate genetic material:
- Creating the Lentivirus: A laboratory procedure to package the gene of interest into a viral vector.
- Infecting Target Cells: The virus is used to infect cells, ensuring the gene of interest integrates into the target cell genome.
Cancer Gene Discovery by Insertional Mutagenesis
- This approach uses insertional mutagenesis to uncover potential cancer-causing genes.
1) Screening Process:
- Insertion Methods: Retroviral infection, transposon mobilization, or lentiviral transduction introduce genetic elements randomly into the genome.
- Tumorigenesis: Mutagenized cells are observed for tumor formation.
- Gene Identification:
- After tumor development, DNA extraction is performed.
- Vector-genome junction amplification allows the identification of insertion sites.
- Sequencing and mapping reveal potential candidate cancer genes.
Validation Process!!
* Transformation Assays: Both in vitro and in vivo assays assess the cancer-causing potential of identified genes.
- Oncogenomic Cross-Species Analysis: Helps to validate findings and identify therapeutic targets and biomarkers.
-Pros:
* Straightforward setup with simple machinery.
* Suitable for in vivo screens and allows incorporation of larger DNA segments.
- Cons:
* Integration-site dependency: Where the vector inserts in the genome can influence results and limit predictability.
What are pros & cons of CRISPR activation systems?
1) Pros:
- Sequence-specific and reversible: CRISPRa can activate native genes without altering DNA sequence permanently.
- Equal representation: Can target genes uniformly across a population of cells.
2) Cons:
- Guide RNA dependency: Requires well-designed sgRNAs for effectiveness.
- Challenges with chromatin: Densely packed chromatin regions can block access to target sites.
- TSS Annotation Requirement: Needs accurate transcription start site (TSS) information for precise activation.
What are pros & cons of gain of function screens?
1) Pros:
- Stable, versatile, and creates dominant phenotypes, making gene function more observable.
2) Cons:
- Non-physiological expression artifacts: Overexpression may not reflect natural gene behavior.
- Size limitations: Large DNA sequences are challenging to introduce using lentiviral packaging, particularly for full-length cDNAs.
Loss of function screens: Gene Knockout (KO) in simple eukaryotes
1) Engineered DNA in Yeast: Uses KO techniques to test gene essentiality.
- Yeast cells can be either haploid, making complete knockouts simpler, or diploid, allowing flexibility in genetic studies.
2) Challenges in Mammalian Cells: Before RNAi and CRISPR, gene knockouts were slow and low-throughput, not practical for systematic or high-throughput studies.
Methods of Loss of Function: inactivation/supression of gene function Screens
o Classical gene knockout
o Insertional mutagenesis*
-> applied in cancer research for tumour suppressor discovery. Knockout of specific genes can reveal those essential for preventing tumour formation.
o RNAi
-> silences genes by degrading mRNA, allowing researches to observe the effects of reduced gene expression on cell function and viability.
o CRISPR knockout
-> cuts DNA at target sites to create gene knockouts.
o CRISPRi
-> Uses dCas9 to block transcription without cutting DNA, creating a reversible gene knockdown.
o Cas13d, Cas 7/11
-> target RNA for knockdown without affecting DNA, providing a versatile approach for RNA-focused loss of function studies.
o mediated gene suppression
—> Other CRISPR systems - Altered Function:
- Base editing: changes individual DNA bases without creating double-stranded breaks (DSBs) (DSB mediated)
- Prime editing: allows for precise edits, adding or deleting small sections of DNA, ideal for more accurate gene modifications.
RNAi pathways
- RNAi interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules.
- dsRNA is a trigger for RNAi.
- Dicer, an enzyme, cuts the dsRNA into small fragments called siRNA or miRNA.
- These siRNA/miRNA molecules are then incorporated into a protein complex called RISC, it uses RNA as a guide to bind to complementary mRNA sequences, leading to the degradation of mRNA or the inhibition of translation, preventing protein synthesis.
1) Endogenous Pathways
- RNAi mechanisms that occur naturally within the cell, often for regulating gene expression.
2) Exogenous Pathways
- Triggered by external sources of RNA, such as:
- Viruses or transposons
- Repetitive DNA sequences or bidirectional transcription
- Aberrant RNA (unusual RNA that could trigger silencing)
Types of RNAi molecules
- shRNA (short hairpin RNA): Delivered via viral or plasmid vectors to silence specific genes.specific genes.
- Synthetic siRNA (small interfering RNA): Chemically synthesized RNA duplexes used to silence genes transiently.
- miRNA (microRNA): Small RNA molecules naturally occurring in cells that regulate gene expression post-transcriptionally.
What are the advantages and disadvantages of different types of RNAi chemistry?
1) Long dsRNAs:
- Simple, cheap, renewable
- Works only in insect and worm cells
2) esiRNA:
- High on-target specificity and silencing efficacy
- Transient, require transfection, hard to make.
3) siRNA (synthetic sgRNA oligos):
- Simple, can be modified for increased stability, single or pooled per gene.
- Transient, require transfection, expensive.
4)shRNA hairpin in retro- or lentivirus (sgRNAs in viral vectors):
- Stable integration or transient transfection, inducible, inexpensive, renewable, pooled screens
- Requires viral packaging step for stable integration.
- Not all sRNAs are as affective (important to check the KD (efficiency of knockdown) at the RNA and at the protein level) to check that it is working.
Why use multiple RNAi vectors?
1) Penetrance of phenotype
- consistent knockdown effect.
2) Off-target effects
- siiRNA#1 against gene A, inhibits Gene A, inhibits gene X then effects phenotype.
What are types of off-target effects?
1) Silencing through partial complementarity
- RNAi molecules can unintentionally bind and silence non-target genes with similar sequences.
2) Immune Reponses
- Foreign RNAi molecules may trigger an immune response, causing cellular stress or inflammation.
- Saturation of the endogenous RNAi machinery
- Excessive RNAi molecules can overwhelm cellular RNAi systems, disrupting normal gene regulation.
How do you mitigate these off-target effects?
- Sequence-dependent effects are cause by the sequence of RNAi.
- Sequence-independent effects are caused by the delivery method.
- If “saturation of the endogenous RNAi machinery” which is sequence-independent, use effectors at the lowest possible concentration, use negative control effectors for comparison.
- If “immune response” and sequence-independent, use effectors at the lowest possible concentration, if sequence-dependent, avoid known stimulatory motifs, use chemically modified effectors, use multiple effectors to confirm phenotypes.
- If “silencing of unintended targets through partial complementarity” and it is sequence-dependent, use effectors at lowest possible concentration and use multiple effectors to confirm phenotypes.
Experimental Controls for RNAi Studies
- Sequence-dependent Off-Target Effects (OTEs): Unintended effects due to partial complementarity to non-target genes, similar to miRNA effects.
1) Solution 1 (Redundancy): Use multiple siRNA or shRNA sequences targeting different parts of the same gene to confirm phenotype specificity.
2) Solution 2 (Rescue): Introduce a resistant version of the target gene to restore function and confirm that reversing the RNAi effect rescues the phenotype.
- Sequence-independent Off-Target Effects (OTEs): Unintended responses that don’t rely on sequence, like immune responses (e.g., interferon).
1) Solution: Use ‘scrambled’ or ‘non-targeting’ control molecules to ensure effects aren’t due to nonspecific activation.
–> Example: In validating MED12 as a crizotinib resistance gene, redundancy and rescue were used to confirm phenotype specificity.
Arrayed Screen
1) Process:
o Individual wells in a culture plate contain chemical compounds and transfection reagents, including shRNAs or siRNAs for RNAi.
o RNAi effectors are arrayed in separate wells (one target per well), cells are added, and phenotypes are assayed individually.
o Cells can be transfected with synthetic siRNA, plasmid-expressed shRNA, or viral-packaged shRNA.
2) Measurements:
o Cellular phenotype is measured using a plate reader or high-throughput microscope.
o Common assays include protein localization, cell size, morphology, internalization, cell viability (luminescent assay), wound-healing assays, fluorescent reporter assays, and imaging.
o In assays with transcriptional activation, a luciferase reporter will indicate activity (on/off) based on transcription levels.
