Functional genomics: gene knockouts, RNAi, transposon mutagenesis

Functional genomics: gene knockouts, RNAi, transposon mutagenesis

1. Functional Genomics:
Unlocking Gene Function
Welcome to this deep dive into Functional Genomics, the field
dedicated to understanding the relationship between an organism's
genome and its observable traits. We will explore key techniques that
allow us to precisely manipulate and study gene function.

2. Chapter 1: The
Foundation
Introduction to Functional Genomics: What and
Why?
Functional Genomics aims to determine the function of every gene product, whether protein or RNA, in an organism's genome. Unlike structural
genomics, which focuses on mapping and sequencing, functional genomics explores the 'how' and 'why'.
Understanding gene roles in complex biological pathways.
Identifying potential drug targets and therapeutic pathways.
Exploring genetic variation and its impact on phenotype.

3. Chapter 2: Targeted
Manipulation
Gene Knockouts: Precisely Silencing Genes
Gene knockout is a technique where a specific gene is inactivated, or 'knocked out', often by replacing it with an artificial
piece of DNA. This allows researchers to study the effect of the gene's absence on the organism's phenotype.
The Principle
By eliminating the expression of a single gene, scientists can
deduce its normal biological function. This method is
fundamental in creating models for human diseases and
studying essential genes.
Key methods include homologous recombination and newer,
more efficient tools like CRISPR-Cas9.

4. Bacterial and Fungal Knockouts: Techniques and
Impact
Generating gene knockouts in microorganisms like bacteria and fungi is crucial for understanding microbial pathogenesis,
metabolism, and resistance mechanisms. These techniques leverage their inherent genetic systems.
Bacterial Knockouts
Often utilise specialised vectors for homologous
recombination. Applications include developing
attenuated vaccine strains and optimising strains for
industrial production of valuable compounds.
Fungal Knockouts
Techniques frequently rely on high rates of homologous
recombination. Used to study virulence factors in
pathogenic fungi and to improve enzyme production in
industrial yeasts.
The precision of gene knockout technology enables researchers to dissect the complex genetic circuitry that governs
microbial life and interaction.

5. Chapter 3: Post-Transcriptional
Control
RNA Interference (RNAi): Silencing Genes with
RNA
RNAi is a natural process in eukaryotes that regulates gene expression by targeting specific mRNA molecules for degradation or translation repression. It acts as
an adaptive immune system against viruses and regulates development.
dsRNA Introduction
Double-stranded RNA is introduced into the cell.
Dicer Cleavage
The Dicer enzyme processes dsRNA into shorter
fragments (siRNA).
RISC Assembly
siRNA integrates into the RNA-Induced Silencing
Complex (RISC).
Targeting mRNA
RISC guides the complex to complementary
mRNA for cleavage, silencing the gene.

6. siRNA and shRNA: Tools for Gene Regulation in
Microbes
While RNAi is naturally prevalent in eukaryotes, synthetic small interfering RNA (siRNA) and short hairpin RNA (shRNA) are
powerful tools used to manipulate gene expression, even in organisms not naturally equipped with strong RNAi pathways.
siRNA (Small Interfering
RNA)
Short, double-stranded RNA
fragments, typically 20-25
nucleotides long, directly introduced
into the cell. They are instantly
incorporated into RISC for
immediate silencing.
shRNA (Short Hairpin
RNA)
Single RNA strand that forms a tight
hairpin loop. Usually expressed from
a plasmid, it is processed internally
by Dicer into siRNA, offering longer-
lasting gene suppression.
Microbial
Applications
Used in fungi and some bacteria (via
heterologous expression) to study
gene function, particularly those
involved in antibiotic resistance and
metabolic engineering.
The ability to transiently (siRNA) or stably (shRNA) suppress gene function provides a flexible approach distinct from permanent
gene knockouts.

7. Chapter 4: Broad-Spectrum Screening
Transposon Mutagenesis:
Randomly Disrupting Genes
Mechanism of Action
Transposons, or "jumping genes," are DNA segments that can
excise themselves and reinsert elsewhere in the genome.
Transposon mutagenesis uses this property to insert a marker
gene randomly into the host genome.
Creating Mutations
When the transposon inserts into a functional gene, it disrupts
the gene's sequence, often leading to a loss-of-function
mutation. This creates a library of random mutants for
phenotypic screening.

8. Types of Transposons and Their Genetic
Footprints
Transposons are classified based on their mechanism of movement. Understanding these types is crucial for controlled
mutagenesis studies in genomics.
Class I:
Retrotransposons
Move via an RNA intermediate, using a "copy-and-paste"
mechanism. These are typically found in eukaryotes and leave a
copy at the original location.
Class II: DNA
Transposons
Move directly as DNA via a "cut-and-paste" mechanism. They
are common in prokaryotes (bacteria) and are essential tools for
microbial functional genomics.
The genetic footprint (the sequence flanking the insertion) allows researchers to rapidly identify the disrupted gene.

9. Random Insertion Mutagenesis: Applications in Discovery
Random mutagenesis provides an unbiased approach to functional screening, especially useful when specific gene functions are
unknown. It is a workhorse in microbial genetics.
Creating Mutant Libraries
Generating thousands of unique
bacterial or fungal strains, each with a
single, random gene disruption.
High-Throughput Screening
Rapidly testing these libraries under
various conditions (e.g., in the
presence of an antibiotic, or high
temperature) to identify strains with
altered phenotypes.
Identification via Sequencing
Sequencing the insertion sites in the
interesting mutants to pinpoint the
disrupted gene and assign a function.
This technique is invaluable for discovering novel genes involved in complex traits like virulence, stress response, and
antibiotic resistance.

10. Chapter 5: Impact
Case Studies: Real-world Impact of Functional Genomics
These techniques are not just theoretical; they drive concrete breakthroughs across various scientific domains, particularly
in the study of microorganisms.
Drug Target
Validation
Using gene knockouts in pathogens to
confirm essential genes for survival,
paving the way for new antimicrobial
drug development.
Metabolic Engineering
Applying RNAi in yeast to enhance the
production of biofuels or high-value
pharmaceutical compounds.
Crop Improvement
Utilising transposon mutagenesis in
model plants to identify genes
controlling drought resistance and yield
traits.
Viral Research
RNAi screens in host cells to identify
human genes crucial for viral
replication, leading to novel antiviral
strategies.

11. Thank you