L25: investigating the function of genes

• by studying organisms that are naturally mutant of a gene, we can work out what the gene might do
• where no natural mutants exists, we can make our own
• by studying both these types of genes, we can learn how particular mutations can lead to phenotypic changes
• natural mutants are where genetic change alters the phenotype
• the cause of polydactyly is a mutation in the gene. it is when they are born with extra fingers or toes. the normal function of this gene is to ensure that the right amount of digits are formed, to prevent this phenotype.
• while variation in the human genome is common, most of this does not affect the phenotype
• mutations are rare, they are a subset of variation, but do not always affect 'fitness'
• around 4000 (20%) human genes have an unknown function, but most are conserved in animals
• we use genetic techniques in model organisms to find out what a gene does by increasing the rate of a random mutation and select the phenotype of interest, then we copy it and insert it into another organism
• by inserting a copied gene into another organism is called transgenesis
• we share many of our genes with other animals
• model organisms are ones that can be easily raisied 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
• zebrafish, 400 million years diverged and 70% human genes
• drosophila, 600 million years diverged and 44% of humans genes
• mouse, 80 million years diverged and has versions (homologous) of most human genes (92%)
• the dna code is universal, so any dna can be used by any organism, even synthetic DNA
• engineering a multicellular organism by adding a foreign DNA is called transgenesis
• we can use transgenic DNA to understand how genes work, to engineer recombinant proteins or in gene therapy approaches
• modern genetics targets mutations to the DNA 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. an example of this is CRISPR-Cas9
• CRISPR is clustered regularly interspaced short palindromic repeats
• Cas9 = CRISPR associated protein 9
• Label CRISPR-Cas9

  A) Cas9 protein

  B) active sites

  C) RNA complex

  D) complimentary sequence that can bind to a target gene

  E) 5'

  F) 3'

  
• The Cas9 enters the nucleus and finds the target sequence in the genome that matches th guide RNA
• Cas9 makes double stranded break in DNA at the target site

  A) cytoplasm

  B) nucleus

  C) Cas9 active sites

  D) 5'

  E) 3'

  F) 3'

  G) 5'

  H) cut

  
• in the absence of a template, DNA repair enzymes to try 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
• if the repair template is provided, it is possible to use this to 'edit' the DNA sequence at the cut site
• Gene editing in somatic targets the cells or organs affected. It does not affect the next generation.
• Gene therapy example: cystic fibrosis: one of the most common lethal single gene genetic conditions, defect in the CFTR gene, which codes for a chloride ion transporter
• Gene editing example: CRISPR-Cas9: sickle cell disease: mutation in the haemoglobin, the oxygen carrying protein in red blood cells, is replaced with a normal version
• Cystic fibrosis

  Delivering DNA with a functional copy of CFTR gene to lung epithelial cells via nebulizer. Extra copy makes good CFTR protein, restoring function to some cells
• Germaline: 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 and before implantation, and can only have healthy embryos implanted
• three parent babies where the faulty gene is on the mitochondrial DNA, nuclear transfer to a donor egg can be used.