D1.3 Mutations & Gene editing Flashcards
Structural changes that cause gene mutations - 1. substitution
One base in the coding sequence of a gene is replaced by a different base
Consequences
- Same sense mutations
Changes a codon for an AA into another codon for the same AA. Does not affect phenotype. - Nonsense mutations
Changes a codon for an AA into a STOP codon. Translation stops before the polypeptide is completed, protein does not function properly. - Mis-sense mutations
Changes a codon for an AA into a codon for another AA.
If the new AA has similar structure or not in a critical position of the protein, not critical change. If the new AA plays a critical role / in a critical position, can have serval and lethal effects. (eg sickle cell anaemia) - Single-nucleotide polymorphisms (SNPs / snips)
A variation at a single position in a DNA sequence.
SNPs can help explain differences in susceptibility to a wide range of diseases across a population.
Structural changes that cause gene mutations - 2. Insertion
Nucleotide is inserted so there is an extra base in the sequence of the gene.
Requires a break to be made in the sugar-phosphate backbone.
Consequence
1. Frameshift mutation –> loss of polypeptide function (view card on frameshift mutation for more)
Structural changes that cause gene mutations - 3. Deletion
Nucleotide is removed, so there is one less base in the sequence of the gene.
Requires 2 breaks to be made in the sugar-phosphate backbone.
Consequence
1. Frameshift mutation –> loss of polypeptide function (view card on frameshift mutation for more)
Frameshift mutation consequences
Loss of function in a polypeptide
- insertions and deletions changes the reading frame for every codon from the mutation onwards, for both transcription and translation
*insertions and deletions of a multiple of 3 are not frameshift mutations
- will still have severe consequences because there is one or more less AA produced and this results in radical changes in protein structure that can affect its functions
Causes of gene mutations
- Mispairing during DNA replication
- When base pairing errors are made and not corrected during DNA rep - Exposure to mutagens
- Radiation: radiation with high enough energy to cause chemical changes in DNA increases the mutation rate. (Eg: UV B & C, X-rays, Gamma rays)
- Chemicals: can also cause chemical change in DNA. (Eg: polycyclic aromatic hydrocarbons PAHs & nitrosamines in tobacco)
Randomness of gene mutations
Mutations are described as random changes
- there is no natural mechanism for changing a base
- organisms have limited control over their overall mutation rate
- can occur anywhere in the base sequences of a genome although some bases have a higher probability of mutating than others because some chemical changes happen more easily
- position of base also affects chance of mutation due to differences in coding and non-coding DNA sequences
Consequences of mutations in a germ cell vs somatic cell
Germ cell: A mutation in a gamete can be passed to the offspring. In most cases, it is a genetic disease.
Somatic cells : Limited consequences because mutations are eliminated when the individual cell dies.
Mutation as the main source of genetic variation. & its link to natural selection
Mutations changes the base sequence of a gene, changing it from one allele into another
–> increases the number of different alleles of genes in a population –> increased genetic variation
Even though mutations are harmful most of the time, still essential to maintain genetic variation and make natural selection possible –> allowing evolution
Natural selection will favour newly formed alleles that help an organism’s survival and select against harmful alleles.
Gene knockout as a technique for investigating gene function
- It is possible to predict which base sequences in a genome are genes by characteristic base sequence patterns. They are known as Open Reading Frames (ORFs). RNA transcribed from them have the start codon AUG, a sequence of triplets, followed by stop codon.
- The gene is removed from genome or made unusable to deduce its function.
Mice as original model organism. Now there are other model organisms like yeast.
- helps us understand diseases
- helps understand the mechanism of drugs
There is a library of knockout organisms available for some species used as research models.
Using CRISPR and Cas9 in gene editing
CRISPR = clustered, regularly interspaced, short palindromic repeats
- the repeats are grouped in one part of the genome
- the number of base pairs in the repeat is between 23-47
- the repeats are separated by other base sequences of a similar length known as spacers
- it is not truly palindromic but the name has stuck
CRISPR gene editing is based on a natural system that exists in many species of prokaryotes.
For appreciation: CRISPR gene editing in prokaryotes :)
- Guide RNA is made by transcribing one spacer and one repeat from a CRISPR array. The spacer forms a variable short base sequence at the 5’ end of the guide RNA (gRNA). It is complementary to the target DNA that is being searched for.
- The enzyme Cas9 can find specific DNA base sequences in the genome, using the gRNA that is bound to it.
- The repeat forms other parts of the gRNA, which are partly double-stranded, generating loops and a distinctive molecular shape that promotes binding to Cas9.
- Cas9 moves along DNA molecules, uncoiling them and bringing the DNA adjacent to the variable base sequence of the gRNA.
- Within its structure, Cas9 contains 2 endonucleases. If the target sequence is recognised, the endonuclease cut one sugar-phosphate bond in each of the DNA strands.
- This makes a double-strand break in the target DNA, destroying the foreign DNA within their cells.
Principles and applications of gene editing
gene editing involves searching for a target base sequence in the genome that is the target and replacing it with the desired sequence.
“search & replace”
potential applications:
1. crop plants
2. eliminate genetic diseases
Prime editing - a modified Cas9-CRISPR system that can make changes to the sequence after it is found
pegRNA
- Prime editing guide RNA (pegRNA) is prepared
- At its 5’ end it has the usual guide sequence transcribed from the spacer in the CRISPR array, together with the base sequences that allow binding to Cas9.
- two extra sequences are added immediately adjacent to each other at the 3’ end of the pegRNA - a primer binding site & an RT template (RNA copy of the desired base sequence to replace the gene being edited)
A version of Cas9 is made with 2 modifications for prime editing
- A reverse transcriptase enzyme is attached to it that can assemble a strand of DNA nucleotides complementary to the base sequence of the RT template.
- One endonuclease in Cas9 is inactivated so it makes a nick in one of the DNA strands and not both.
Process of Prime Editing using Modified Cas9 and pegRNA in prime editing
- Modified Cas9 and pegRNA are introduced into cells where they form a complex and begin the process of gene editing
- Cas9 moves along the DNA molecule, searching for the target sequence. The target sequence is identified using the guide sequence at the 5’ end of the pegRNA.
- Where the target sequence is recognised on one of the two strands, a nick is made in the complementary sequence on the other strand. This creates a 3’ and 5’ end in that strand.
- The two DNA strands are separated, with the gRNA linked to the target sequence by base pairing.
- The primer binding site of the pegRNA binds by base pairing to the other DNA strand, on the 3’ side of the nick. This creates a site at which reverse transcription can be initiated.
- Reverse transcriptase adds DNA nucleotides one by one to extend the DNA strand from the 3’ end created where the DNA was nicked.
- The nucleotides are added from 5’ to 3’ direction, using RT template to determine the base sequence.
- The two strands of DNA pair up again. The sequence assembled by reverse transcriptase displaces the sequence that it is replacing. The replaced sequence becomes a single-stranded flap.
- Nucleotides of the single-stranded flap are removed, editing the original base sequence out.
- There will be some mispairing of bases where the double-stranded DNA has reformed, because of difference between the replacement and original base sequences. DNA enzymes correct the mispairing such that the unedited strand becomes complementary to the edited strand.
Conserved & highly conserved gene sequences
Conserved sequences: identical or nearly identical across a species or many species
Highly conserved sequences: identical or nearly identical over long periods of evolution.
Many conserved sequences have specific functions
1. in protein-coding elements of the genome
2. occur in elements that are transcribed to make rRNA or tRNA
3. sequences used to regulate gene expression
Non-coding elements are also conserved in many organisms
- their functions are unknown but based on the functional hypothesis, they probably do have functions
- hypothesis 2: the conserved non-coding elements might be in regions of the genome where mutation rates are low
2 hypotheses to account for conserved & highly conserved gene sequences
- Functional requirements for the gene products.
Any mutations created would not have been passed down because the organism would not have been biologically fit enough to reproduce.
When speciation occurs, both resulting species could inherit these sequences even when groups diverge in their traits. –> explains conserved sequences.
- Lower mutation rates in non-coding gene sequences.
Mutation rates are not identical throughout the genome.
Eg: HACNS1 gene is highly conserved across a wide range of birds & mammals, implying that it has been conserved over millions of years. –> explains highly conserved sequences
