D1.3 Mutation and gene editing

gene mutation

• when changes occur to the base sequence of a gene

• random, NOT the same as deliberate changes made by scientists in gene editing

types of gene mutation

• substitution

  • replacing a base with another base in the coding squence

  • happens by chemical changes to bases / mispairing during DNA replication

• insertion

  • inserting extra nucleotide in the coding sequence

• deletion

  • removing a nucleotide from the coding sequence

types of substitution mutations

• same-sense mutation: base substitutions that change one codon for an amino acid into another codon for the SAME amino acid.

• non-sense mutation: base substitutions that change one codon for an amino acid into a stop codon, terminating translation process

• mis-sense mutation: base substitution that change one codon for an amino acid into another codon for another amino acid

mis-sense mutations effects

• new amino acid has similar structure to original amino acid → not much effect

• small proportion → improves function of protein, increase individual’s chance of survival

  • lactose tolerance mutation: natural selection favoured those with lactose tolerance gene

• leads to formation of new alleles of a gene and increases genetic diversity

frameshift mutations

change the reading frame for every codon from mutation onwards

mutation example

BRCA1 gene - a tumour suppressor gene

• BRCA1 protein (DNA repair)

  • Mending double strand breaks

  • Correcting mismatches in base pairing.

• mutation of BRCA1 gene → increased risk of mutation → increased risk of tumour formation and cancer

mutagens

• radiation

  • high frequency radiation = high energy to cause chemical changes

• chemicals

  • mutagenic chemical substances causing chemical changes in DNA

  • examples

    • polycyclic aromatic hydrocarbons

    • nitrosamines

randomness of mutation

1. Mutations are random changes and unpredictable.

2. The consequences of mutations does not influence the probability of its occurrence. i.e. mutations intended to be beneficial do not exists.

3. mutations can happen anywhere in a sequence of a genome but some bases have a higher probability of mutating than others

   • since mutations are chemical changes and certain chemical changes happen more easily than others

mutation in somatic vs germ-line cells

mutation in somatic cells → will be eliminated when individual dies; mutation in germ-line cells → mutations will be passed onto gametes

proto-oncogenes vs oncogenes

proto-oncogenes: genes with roles that control cell cycle and division

oncogenes: proto-oncogenes with mutations, cancer-causing genes

mutation variation and evolution

• leads to speciation and evolution

• only a small proportion of population will have characteristics that allow them to adapt to environmental change

• no mutation = no genetic variation = no adaptation to conditions in changing environment = higher chances of extinction

process of gene knockout

1. DNA (that can be inserted into embryonic mouse cells’ genome as replacement for a target gene) is prepared → deletes a copy of the target gene

2. successful cells from this procedure are selected and grown into adult mice. these mice will only have 1 copy of the target gene

3. females and males mated → 25% of offsprings expected to have no copies of target genes (referred to as knockout mice) (genes made inoperative)

4. phenotype of these knockout mice is investigated to find out which traits have been changed by deletion of the target gene

function of gene knockout

• discover function of genes

• predict which base sequences in a genome are genes

example of gene knockout

PIEZO2 gene

• PIEZO2-knockout mice urinate less frequently than normal individuals

• PIEZO2 gene found to code for a mechanosensitive ion channel that acts as a pressure sensor in bladder

  • humans who naturally lack PIZEO2 gene also show impaired bladder control

gene editing

• inserting/deleting/substituting bases to generate desired sequence

• CRISPR-Cas9 = gene editing system

successful example of CRISPR-Cas9

• treating sickle cell anaemia

  • people with sickle cell anaemia have RBC that cannot transport oxygen efficiently

  • scientists used CRISPR to edit a gene in the cells of a patient’s bone marrow → such that patient would produce foetal haemaglobin

    • foetal haemaglobin → higher absorption of oxygen → reduced fatigue and breathlessness in patient

Cas9

• found in bacteria

  • destroys viral DNA when it enters bacterial cell

• endonuclease enzyme

  • separates strands of DNA

  • finds target sequence in DNA using guide RNA (gRNA)

  • cuts DNA at a target sequence

guide RNA

• synthesised by transcribing “spacer” and “repeat” from a bacterial genome - CRISPR array

  • spacer: complementary to target sequence

    • at the 5’ end of gRNA

  • repeat: binding site for Cas9

    • partly double-stranded

    • generates loops and distinctive molecular shape

CRISPR Cas9 process

Cas9 used in gene editing:

• only cuts 1 strand of DNA - not both

• has reverse transcriptase attached - makes a strand of DNA with base sequence complementary to an RNA template

gRNA used in gene editing:

• has a template sequence used by reverse transcriptase

• has a primer binding site, same base sequence as DNA adjacent to target sequence

stages in gene editing:

1. gRNA binds to Cas9

   • gRNA has bases complementary to that of target sequence

2. Cas9 separates 2 DNA strands, moves along DNA molecule to find target sequence using spacer of gRNA

3. complementary base pairing between target DNA and spacer → starts gene editing process

4. spacer bound to target sequence, Cas9 makes a nick/cleave on the other strand → creating 3’ and 5’ ends

5. DNA strand on 3’ end of the nick links to primer binding site on gRNA by complementary base pairing

6. reverse transcriptase adds DNA nucleotides to the 3’ end using template sequence in gRNA to determine the base sequence

7. gRNA detaches from Cas9, 2 DNA strands pair up again, sequence assembled by reverse transcriptase displaces original sequence, which becomes a single-stranded flap

8. nucleotides in the flap removed → edits out the original base sequence