Lecture 25 ( investigating the function of individual genes)

Question 1

How do we get information about the function of a gene from its phenotype?

Answer 1

By studying organisms that are naturally mutant for a particular gene, we can work out what that gene might do

Where no natural mutants exist we can make our own

By studying both these types of mutants we can learn how particular mutations lead to phenotypic changes

Question 2

Mutant

Answer 2

A cell or organism carrying an altered or mutant allele.

Question 3

Natural mutant

Answer 3

Natural mutants - where genetic changes alters the phenotype - gives clues to gene function

The phenotype is what we see. The cause of a particular phenotype is a mutation in a gene. The normal role of a gene is to prevent the phenotype in this case.

Question 4

The value of mutant

Answer 4

While variation in the human genome is common, most of this does not affect phenotype

Mutations are rare, they are a subset of variation, but does not always affect “fitness”

Around 4500 (23%) of human genes have an unknown function. Luckily many of these are conserved in other animals - even in many cases with fruit flies and yeast! If we can find or create mutants in these related genes, we may learn their functions in humans

Question 5

Mutations are usually named

Answer 5

After the mutation it causes

Question 6

How do we use genetic techniques to find out what a gene does?

Answer 6

Study raises are that are naturally mutant for that gene are rare.

Increase the rate of random mutation, select for a phenotype of interest and sequence the genome to identify the mutation (genetic screen)

Take a gene you are interested in, copy it and insert it into another organism (transgenesis/genetic engineering)

Deliberately break a particular gene to see what happens (targeted mutation/gene knockout/reverse genetics)

This type of approach is called functional molecular genetics

Question 7

Model organisms

Answer 7

Model organisms can be used to make mutants.

We share many of our genes with other animals. Model organisms are ones that can be easily raised in a controlled environment and are easy to manipulate genetically.

Each organism has a different approach that works best for making changes to the DNA genome

Mose has versions of most human genes (92%), zebrafish (70%) and drosophila (44%)

Question 8

How can mutants be made?

Answer 8

Mutants can be made by treatment of gametes with mutagens such as X-rays or chemicals

Mutations made this way are random and can give quite dramatic phenotypes

Question 9

Transgenesis

Answer 9

The process used to create transgenic organisms.

Transgenesis is the process of introducing a gene (referred to as a transgene) from one organism into the genome of another organism. The aim is that the resulting transgenic organism will express the gene and exhibit some new property or characteristic.

The DNA code is universal, so any DNA can be used by any organism - even synthetic DNA. Engineering a multicellular organism by adding in “foreign” DNA is known as transgenesis. We can use transgenic DNA to understand how genes work, to engineer recombinant proteins (synthetic biology), or in gene therapy approaches)

There is a regulatory sequence ( this is something that makes it work when you put it into the organism of choice) and then the gene you want to express which you do by adding the coding part of this gene

Question 10

Transgene

Answer 10

a gene which is artificially introduced into the genome of another organism.

Easiest way to introduce a transgene is to put it into a zygote

Question 11

How to find out if a variant is pathogenic?

Answer 11

Sequence genomes

Map to human reference genome (test siblings and parents)

Find common variants and novel variants

Investigate novel variants and see if they are predicted to be benign or predicted to be harmful (how it might change a protein product of a gene an change phenotype)

Validate and test the ones predicted to be harmful

Question 12

How do we know if a gene variant is pathogenic?

Answer 12

Modern genetics targets mutations to the DNA sequence of your choice to ‘break’ specific genes

We can damage or modify the gene we are interested in by genetically modifying an organism or cell line

By examining the organism, or its offspring we should be able to work out what the gene normally does

There are many ways in which to do this but we will look at one in this course known as CRISPR-Cas9

Question 13

Targeted mutation with CRISPR-Cas9

Answer 13

CRISPR = Clustered regularly interspaced short palindromic repeats (effectively molecular scissors) 
Cas-9 = CRISPR associated protein 9 

Evolved in bacteria for antiviral defines

Can decide which gene you wish to mutated

Design a short “guide’ RNA that only binds to your gene of interest (it will be complementary to the gene of interest)

Mix these all together - Cas9-guide RNA complex

A technique for editing genes in living cells, involving a bacterial protein called Cas9 that helps defend bacteria against bacteriophage infections. Cas9 acts together with a ‘guide RNA’ complementary to a gene sequence of interest. In a secondary application, the CRISPR-Cas9 system is being tested in the alteration of genes in insects to eliminate the transmission of insect-borne diseases.

Use it to break genes and you can use it to slightly change genes

Question 14

CRISPR stands for

Answer 14

Clustered regularly interspaced short palindromic repeats

Question 15

Cas-9 stands for

Answer 15

CRISPR associated protein 9

Question 16

CRISPR-Cas9 process

Answer 16

Cas-9 has active sites that can cut DNA

There is guide RNA engineered to ‘guide’ the Cas-9 protein to a target gene. Complementary sequence that can bind to a target gene.

Cas9 enters nucleus and finds target sequence in genome that matches the guide RNA

Cas9 makes double stranded break in DNA at target site. Resulting in a hole in the target gene.

In the absence of a template, DNA repair enzymes try to patch up the cut. This often results in errors as there is no template to read from

Small indels are created at the target site, the gene is potentially disrupted, or mutated (scientists can disable the target gene to study its normal function). If a repair template is provided, it is possible to use this to ‘edit’ the DNA sequence at the cut site (“gene editing”) (Normal functional gene for use as a template by repair enzymes is provided. If the target gene has a mutation, it can be repaired)

Question 17

How can we fix genetic disease in somatic cells?

Answer 17

Targets the cells or organs affects

Does not affect the next generation ( it is not a change to the germline)

Best candidates are single gene diseases caused by a loss of function mutations as you can just add the good version of that particular gene

Gene therapy is an option (put a good copy of the gene into the cells of the affect individual)

Gene editing with CRISPR-Cas9 is also being explored as an option

Question 18

Gene therapy for cystic fibrosis

Answer 18

Delivering DNA with the functional copy of the CFTR gene to lung epithelial cells via nebuliser. Extra copy makes good CFTR protein, restoring the function to some cells.

Liposome holes the normal CFTR gene and is able to directly fuse with the cell membrane and release their contents into the cell. This gene the goes through transcription and translation and then produces the normal protein.

Question 19

How can we fix genetic disease in germline cells?

Answer 19

Have to consider the ethics

Pre-implantation genetic diagnosis - in families with an identified risk, IVF can be used to make embryos from the parents’ eggs and sperm. These embryos can be tested before implantation, and only healthy embryos are implanted

Three parent babies: Where the mother is carrying a mitochondrial disease, the faulty gene is on the mitochondrial DNA, nuclear transfer to a donor egg can be used (Mitochondrial transfer works by replacing the damaged mitochondria in the mother’s egg with healthy mitochondria from another woman’s donor egg. The developing embryo now has nuclear DNA from the mother and father, as well as mitochondrial DNA from the donor egg.)

CRISPR gene ‘edited’ babies : big ethical questions are faced with this before it will ever be used