D1.3 Mutations and gene editing

• Distinguish between base substitution, insertion and deletion mutations

  1. Base substitution is replacing one or more nucleotides for the same number of nucleotides

  2. Insertion is adding one or more nucleotides

  3. Deletion is removing one or more nucleotides
• Frameshift mutation

  Insertions and deletions of multiples of 3 nucleotides add/remove individual amino acids from the chain while those not multiples of 3 change the reading frame
• Base substitution mutation in coding sequences

  May cause changes in amino acid sequences
• No natural mechanism is known for making a deliberate change to a DNA sequence
• Mutagens can lead to mutation by altering complementary base pairing, breaking hydrogen bonds, uncoiling the DNA double helix, separating or breaking strands
• Gene knockout is a technique for investigating the function of a gene by changing it to make it inoperative
• Genetic code degeneracy

  Some base substitution mutations do not cause changes in amino acid sequences due to the genetic code being degenerate
• Outline causes of gene mutation

  1. Errors of polymerase in DNA replication

  2. Mutagens
• Source of new alleles of a gene

  • Gene mutation is the original source of all genetic variation
  • Single-nucleotide polymorphisms (SNPs) are the result of base substitution mutations
• Genetic variation


  Differences in DNA sequence among individuals of the same species
• Single-nucleotide polymorphism

  Individual differences in the DNA base sequence, the result of base substitution mutation
• Distinguish between same-sense, nonsense and missense base substitution mutations


  1. Same sense means the same amino acid is encoded

  2. Nonsense means codon STOP proceeds

  3. Missense means a different amino acid is encoded
• Base substitution mutation in noncoding sequences


  May not cause changes in amino acid sequences
• Distinguish between germ cells and somatic cells

  1. Germ cells are involved in reproduction while somatic cells make up the body

  2. Consequences of a germ cell mutation can cause genetic diseases in offspring, while in somatic cells, it can lead to tumors
• Distinguish between beneficial, neutral and harmful gene mutations

  1. Neutral mutations are the most frequent

  2. Beneficial mutations are rare (e.g., antibiotic resistance in bacteria)

  3. Harmful mutations can cause genetic diseases in offspring or tumors
• Gene mutation is the original source of all genetic variation
• Mutation


  Permanent change in the genetic material, i.e., structural changes to genes at the molecular level
• Mutagen

  Physical (UV, X rays, high temperature), chemical (benzene, formaldehyde), biological (some viruses) agents that can lead to mutation
• Outline the method scientists use to “knockout” genes

  An intentional alteration in the gene sequence using CRISPR to make it inoperative, followed by observing changes in the organism's characteristics to determine the gene's function
• Consequences of insertions and deletions on polypeptide structure and function

  • May cause polypeptides to cease to function
  • Frameshift changes cause a completely different polypeptide chain to be synthesized
  • Major insertions or deletions affect the three-dimensional structure of the protein
• Uses of CRISPR Cas9 gene editing

  • Cancer research
  • Transgenic models in genetics
  • Plant disease resistance
  • Increasing crop yield in plants
• Conserved sequence of DNA are identical or similar across a species or a group of species
• Two hypotheses that account for conserved sequences between species are: 1) slower rate of mutations 2) the functional requirements for the gene product ie. protein structure conditions its functionality
• Use of the CRISPR sequences and the enzyme Cas9 in gene editing
• Mutations to proto-oncogenes and tumor suppressor genes can lead to the development of cancer
• Ethical implications of gene editing

  Scientists across the world are subject to different regulatory systems. There is an international effort to harmonize regulation of the application of genome editing technologies such as CRISPR
• Comparison between rates of cell division and growth and the capacity for metastasis and invasion of neighboring tissues between normal cells and cancerous cells

  Cancerous cells have higher mitotic index values than normal cells
• Gene editing using CRISPR Cas9

  The genome is cut at a desired location to remove existing genes or add new genes. Cas9 is an endonuclease enzyme that recognizes and cuts DNA at a targeted location. A synthetic gRNA sequence is prepared to indicate the cut site, and then the Cas9 enzyme is added to cut between the specific base pairs, adding or removing a specific gene