Term 2 Lecture 9: Genetic mutants and gene editing Flashcards
Creating a mutant is a reverse genetics method
By knocking out a gene to observe the resulting phenotype of the changed genotype the role of the protein the gene encoded for can be observed e.g. knocking out a gene in Arabidopsis causes the plant to not produce sexual organs and produce extra petals in the flowers instead.
There are many ways to create mutants and different types of mutants that can be created
1) knockout mutants ( loss of function) - function of a gene is disrupted
2) knock down mutants (reduction in gene expression) can be tissue specific
3) overexpression mutants (gain of function) - normal regulation disrupted - expression occurs where it does not normally or at a higher level
In model species many gene mutations are mapped to the genome and information is available to researchers in bioinformatic databases that show:
-chromosomal locations
- genes
- transcripts - including splice variants and alternate translation initiation sites
- clicking on areas within the genome database provides specific annotation information on each gene, information on work that has already been done on that gene and different transcription levels
1) knockout mutants
In the past they were created by UV or x-ray radiation nowadays it is normally done by chemical treatment often with alkylating agents e.g. EMS
- insertion mutants are created by:
transposons -genes that move around in the genome causing mutation
T-DNA - can integrate into plant genomes
Short insertions/deletions carried out by dsDNA cuts - gene editing by CRISPR-Cas9 - much more accurate
Transposons: transposable elements cause mutations
transposable elements= sequences that can move about in the genome
transposition: movement of transposons
You can identify where a transposon has relocated to by PCR reactions to amplify the DNA either side of the transposon to find its location.
process of locating a transposon:
1) staggered cuts made in the target DNA
2) transposable element inserts itself into the DNA
3) as transposable elements have blunt ends the staggered cuts leave short areas of exposed ssDNA
4) replication of this ssDNA creates flanking direct repeats
(see Pierce chapter 18.4)
on a bioinformatics database transposon insertion lines are available for all genes in the sequenced genome
1)knockout mutants: Insertion mutants in plants are created by T-DNA
using Agrobacterium vectors containing edited Ti plasmids
edited T-DNA is incorporated into the Arabidopsis cells - this method has been used to create insertion mutants in nearly all genes in Arabidopsis
2) Knockdown mutants (reduction in gene expression)
-Interference RNA (RNAi) is a natural mechanism that blocks translation. Short dsRNA is unwound and binds to complimentary mRNA by a protein complex which also catalyses the breakdown of the mRNA.
-small interfering RNA (siRNA) can be synthesised in the laboratory to inhibit gene expression ( here the DNA isn’t mutated - see Pierce chapter 14.5 and 19.6)
Normally in translation mRNA is produced and translated to give a protein, this can be blocked by siRNA which starts of as dsRNA and unwinds itself to ssRNA able to bind to complementary sequences on mRNA targeting them for destruction so that the protein is not produced.
ds siRNA is produced by using an expression vector, you identify the gene to be regulated then clone the fragment to have promotors at both ends - so that through transcription the same RNA sequence is produced in both directions providing ds siRNA
When produced in a cell siRNA modulates the expression of a particular gene - promotors may be tissue specific or inducible promotors so the si RNA can silence gene expression or modulate/regulate it very precisely to create/ observe the resulting phenotype.
Overexpression mutants (gain of function)
normal regulation disrupted
can be created by insertion of strong promotor/enhancer regions in the genome or by disrupting regulatory regions
- have an important effect as too much of a protein can be as detrimental as too little/ none
Gene editing: CRISPR-Cas9
over the past few years new molecular tools have been developed for site specific cutting of DNA.
The first generation were engineered nucleases e.g. zinc finger nucleases (ZFN) and transcription activator-like nuclease (TALEN) system. They were cumbersome and have now been almost completely replaced by the CRISPR-Cas9 system.
CRISPR-Cas9 is now routinely used in all the model species and several more.
It’s a powerful molecular tool that enables us to make site specific cuts in DNA.
Restriction enzymes (RE) act as a defense mechanism for bacteria protecting them against invading pathogens (primarily bacteriophages.) CRISP-Cas bacterial immunity provides a ‘molecular memory’ of pathogens that have invaded before - they recognise a pathogen on reinfection and specific RE are used to perform dsDNA cuts killing the pathogens before they can harm the bacteria.
CRISPR-Cas9 was discovered by sequencing bacterial genomes and were found in many prokaryotic genomes including archaea.
CRISPR stands for
Clustered
Regularly
Interspaced
Palindromic
Repeats
Between the CRISPR sequences are short palindromic spacers of unique 24-bp fragments of DNA from viruses that previously infected the prokaryotic cell.
The Cas nuclease has 2 domains
Rec (recognition) region that recognises where to cut the target sequence to incorporate into a spacer region
Nuc (nuclease) domain that performs the cut
The original Cas protein is called Cas9 and was isolated from Streptococcus pyogenes however there are many different Cas proteins now used in gene editing with different structures and activities - Cas9 however is still commonly referred to as just Cas.
Cas9 nuclease incorporates unique viral spacer DNA sections so that if the bacteria is reinfected with the same bacteriophage again it is recognised. The spacer region binds to the ‘protospacer’ (homologous region on the virus DNA) on the infecting phage.
The Cas nuclease makes a ds cut in the viral genome resulting in the death of the virus. An effective defence mechanism.
Rec initially binds to a protospacer adjacent motif (PAM - typically NGG) and Cas unwinds the DNA then the spacer region binds to the protospacer and ds cut is carried out.
Single guide RNA (sgRNA) in more detail
and using CRISPR for gene editing
sgRNA is the unique sequence complimentary to the target region (seed sequence)
lots of repetition in this sequence causes it to form many stem and loop structures
this is the region coded for by the palindromic cluster repeats linked by the spacer regions that bind to target sequences (protospacers.)
When a bacteriophage genome undergoes dsDNA cuts the virus dies.
When CRISPR-Cas9 is used to cut bacterial DNA the bacteria naturally carries out non-homologous end joining - emergency repairs carried out by faster and less accurate DNA Pol which often leads to mutations due to causing frameshifts in the open reading frame.
There is another method used for targeted mutation where donor DNA with complimentary ends on either side of the break is introduced to ‘patch’ the break however it acts at a low frequency and is not 100% accurate
Another method known as Prime Editing is being developed to allow precise changes to be made extra reading
The CRISPR-Cas system is then moved from bacteria into an organism - how is this done?
using a CRISPR-Cas9 expression plasmid developed specifically for purpose - these are now readily available commercially.
Vectors must have:
BamH1 and BSmB1 region - produced by PCR or chem synth
RNA region for sgRNA
Strong promotor to drive Cas nuclease gene
Cas nuclease gene
The cassette is then cloned into an appropriate vector e.g. T-DNA for plants, virus for animals or Cas9 and sgRNA can be used directly in a microinjection (for transient transformation)
For model organisms many CRISPR-Cas lines are available
Forming a range of knockout or overexpression lines.
sgRNA sequences are also given so that you know where exactly on your gene of interest the cut is going to be made.
^ This has become the most commonly used mutational system creating or using existing CRISPR-Cas9 and ordering from databases.
Example of CRISPR-Cas use: mouse model
Long et al used adeno-associated virus-9 to deliver the CRISPR-Cas9 gene editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein that is deficient in patients with Duchenne muscular dystrophy.
Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle cells and improved skeletal muscle function.
^Postnatal genome editing partially restores dystrophic expression in a mouse model of a gene coding for dystrophin a muscle protein deficient in patients with Duchenne muscular dystrophy - Long et al (2016) Science 351 pp400-403
Mouse and human dystrophin genes are very similar with slightly different exons. The protein they encode is identical so near 100% homology.
MDX mutation in mouse model is a point mutation on exon 23 converting it to a stop codon and preventing dystrophin protein from being produced.
One method for correction, as used here, is to remove exon 23 to ‘repair’ the open reading frame with minimal effect on the protein produced.
So 2 sgRNAs designed for either side of exon 23 are used to remove it allowing exons 22 and 24 to splice together.
The virus carrier is used to introduce Cas9 protein and 2 sgRNAs for either side of exon 23. It is injected into the mouse muscle inducing transient transformation - to see if they have any effect when introduced.
It was injected into cardiac and skeletal muscle and found to massively increase dystrophin gene expression - showing gene editing was successful.
Mouse muscle strength was tested by ‘grip test’ and found to be greatly improved.
SO the CRISPR-Cas9 tool provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth.
However there are some problems - the major one being that the cutting is ‘random’ within the target region and hosts repair mechanisms introduce errors.
A new technique, prime editing, is being developed to make sure that the cut occurs in a precise region and not randomly
Ethics of gene editing
Important to consider - designer pets and babies etc
Plans to bring species such as the dodo ‘back from the dead’
