D1.3 Mutation and Gene Editing

Mutation Definition

A mutation occurs when the sequence of DNA bases is altered.

3 main types of gene mutation

Substitution:

• one base in coding sequence is replaced by another.

• can happen by chemical changes to bases or by mispairing during replication.

Insertion:

• a nucleotide is inserted, so extra base in the sequence.

• more major as a break is needed in the sugar phosphate backbone.

Deletion:

• a nucleotide is removed so there is one less base in the sequence.

• this requires two breaks in the backbone.

Consequences of base substitution:

Same-sense

Nonsense

Mis-sense

Mostly neutral or deleterious and in some cases lethal. In non-coding DNA, they are unlikely to have any effect – only changes to coding sequences can affect amino acid sequences of polypeptides

Same-sense

Same-sense mutations change one codon for an amino acid into another codon for same amino acid. Possible due to redundancy of genetic code. Eg. a change from AGC to AGT still codes for serine. No effect on phenotype

Nonsense

Nonsense mutation change a codon for an amino acid into a stop codon (ATT, ATC or ACT). So translation is terminated before polypeptide is complete. Doesn’t function properly.

Mis-sense

Mis-sense mutations alter one amino acid in the sequence in a polypeptide. May not have much effect if new amino acid has similar structure + chemical prop. But can be lethal, cause of many genetic diseases like sickle cell

Mis-sense further info

When DNA from individual humans is sequenced, large numbers of historical base substitutions are found.

Known as single-nucleotide polymorphisms (SNPs).

Can occur in non-coding regions of DNA.

Presence of some SNPs is associated with certain diseases.

These correlations allow scientists to determine an individual’s genetic predisposition to developing a disease.

Consequences of Insertions and Deletions

• Major insertions and deletions almost always result in a functionless polypeptide.

• Minor insertions and deletions (one or two nucleotides) can have the same result. 

• They are frameshift mutations – they change the reading frame of every codon from the mutation onwards. (→)

Causes of Gene Mutation

• Increased risk during DNA replication, when base-pairing errors are sometimes made and not corrected by DNA repair.

• Radiation:

  - increases rate if it has enough energy to chemically

    change DNA

  - alpha particles, gamma rays, x-rays & UV are mutagenic.

• Chemical: 

  - chemical substances can cause chemical changes to DNA

  - eg. polycyclic aromatic hydrocarbons & nitrosamines.

Randomness in Mutation

• Unpredictable and cannot be directed by living organisms to achieve an intended outcome.

• Mutations can occur anywhere in a genome’s base sequence, although some bases have a higher probability of mutating than others.

• This is because some chemical changes happen more easily.

• Effects of a mutation in a somatic cell may be tested when the gene is expressed, but it is eliminated when cell dies. 

• Traits due to mutations acquired during a lifetime which prove to be beneficial cannot be inherited by offspring.

• This helps to explain how evolutionary change occurs.

Consequences of mutation in germ & somatic cells:

• Germ cells give rise to gametes, so genes in germ cells can be passed to offspring.

• So, a new allele, produced by a mutation in a germ cell, can be inherited.

• causes a genetic disease.

• important to minimise mutation in germ cells

• Mutations in somatic cells are eliminated when the individual dies, so consequences are limited.

Mutation as a source of genetic variation:

• Allele = variant of a gene.

• Mutations change an allele into another allele.

• Mutations increase number of alleles, and therefore genetic variation, in a population.

• Meiosis and sexual reproduction can increase variation by generating new combinations of alleles, but mutation is the original source of all genetic variation.

• Most mutations are either neutral or harmful however mutation is needed in all species.

• Natural selection requires genetic variation, so species cannot evolve without it.

Gene Knockout

Gene knockout = technique used to investigate function of a gene by changing it to make it inoperative.#

Method:

1. Prepare DNA with a base sequence that allows it to be

     inserted into genome of embryonic mouse cells as a

     replacement for a target gene, which is deleted.

(2) Select successful cells and grow into adults – these will

     only have one copy of target gene. 

(3) Mate males and females – 25% of offspring will have no

     copies of target gene, ie. knockout mice.

(4) Investigate phenotype to find out which traits have been 

      altered. 

Use of CRISPR sequences & Cas9 in gene editing:

• CRISPR = Clustered, Regularly Interspaced, Short Palindromic Repeats.

• Repeats – same base sequence occurs several times.

• Short – number of base pairs in repeat is 23 – 47.
  

• Clustered – repeats grouped in one part of genome.
  

• Regularly interspaced – repeats separated by spacers. Each spacer is unique. Derived from viruses and allow viral DNA sequences to be recognised if reinfection occurs.
  

• Palindromic – each repeated sequence has parts which read the same backwards as forwards (dyad symmetry). 

Cas9

• Finds specific base sequences in genome using guide RNA (gRNA) bound to it. Made of a spacer and repeat

• Spacer forms a base sequence at 5’ end of gRNA which is complementary to target DNA being searched for. 

• Repeat forms other parts of gRNA generating loops and distinctive molecular shape that promotes binding to Cas9. 
  

• Cas9 moves along DNA molecule, uncoiling it and bringing DNA adjacent to variable base sequence of gRNA.
  

• Cas9 contains two endonucleases - if target sequence is recognised, endonucleases cut one sugar-phosphate bond in each strand forming double-strand break in target DNA.
  

• This how prokaryotes recognise & destroy foreign DNA – especially viral DNA.

Using Cas9-CRISPR in gene editing:

• Cas9-CRISPR can be used to find target sequence. It has been modified so that it can also make changes to it.

• Prime editing is a promising approach.

• Prime editing guide RNA (pegRNA) is prepared.

• At 5’ end, it has usual guide sequence transcribed from the spacer in a CRISPR array, along with sequences that allow binding to Cas9.

• Two extra sequences are added immediately adjacent to each other at 3’ end: