Genetic Manipulation Technology 5&6 Flashcards
dodName 3 methods of genome
editing
Zinc finger nucleases
Talens
Cas9/CRISPR
What do these genome editing methods actually do
They find ways to direct a DNA cleaving enzyme to specific sites in any genome where you want to make mutations
What are zinc-finger nucleases
Protein motif that binds to 3’ base end - every zinc finger has a codon that is specific to each 3 base pairs - a chain of base pairs will bind to specific sequences and transcribed.
-> Zn-finger transcription factor binds specific consensus DNA sequences around genes and activates transcription. Different Zn-finger transcription factors with different DNA binding specificities control the regulation of different
What does FOK1 do
A single-stranded DNA nuclease - FOK1 cuts the DNA strand and the ZFN cuts the other side (done on both strands) –> creates a double-stranded DNA break
What is non-homologus end joining (NHEJ)
Repairs by DNA enzymes will usually cause errors - causing point mutations - 70-80% of time (insertions or deletions)
Why are ZFN good
Work on any animals
Work in vitro and vivo, with good specificity
What are TALENS
Transcription Activator-Like Effector Nucleases
How do TALENs work
Work similar to ZFNs
comprise of nonspecific FOK1 nuclease domain and customizable DNA binding domain on both sides to cut double stranded DNA
What do TALENs look like
33-35bp repeats - easily recognisable
How do TALENs cause mutations
Non-homologous end joining
step by step:
1. 2 talens TALENs binding specific sequence of target gene to create double strand break
2. 2 hypervariable amino acids determine which base each TALE repeat binds to
3. A TALE binding its target DNA sequence
What is Cas9
Double-stranded endonuclease - cuts DNA on both strands with one cas9
Where does Cas9 come from
Bacterial acquired immunity against virises
What is CRISPR
Clustered regularly interspaced short palindromic repeats - essentially means equally spaced repeats of DNA bases with coding DNA in between related to pathogens. Used in bacteria to identify bacteriophages/viruses in the past. When this coded DNA is identified from the pathogen it is matched up with Cas9 –> cleaved and removed to allow cas-mediated mutagenesis to occur thus killing the pathogen.
What does Cas-mediated mutagenesis stand for
CRISPR associated mutagenesis
What are the two parts of Cas-mediated mutagenesis and their roles
CRISPR-RNA
Transactivation CRISPR-RNA
These RANA’s guide Cas9 to the specific site to cut gene and mutation is caused through repair
Where can Cas-mediated mutagenesis only occur
Needs to be adjacent to a protospacer adjacent motif (2-5bp that are NGG)
How does cas-mediated mutagenesis/CRISPR work
1,The first CRISPR crRNA is complementary to part of the target gene, upstream of the PAM.
2.Go into DNA duplex and binds by base-pairing
3. The transactivating CRISPR tracrRNA () has a region that is complementary to the rest of the crRNA
4.It binds by base-pairing
5. A Cas9/crRNA/tracrRNA complex binds the target sequence
6.Cas9 will make a double-stranded break in the target DNA, a variable number of bases from the PAM. Break will be ragged, with single strand overhangs
BUT….
If you introduce Cas9 and crRNA and tracrRNAs into eukaryotic cells, it will work to cleave the targeted DNA
But rather than killing the cell, DNA repair enzymes will try to repair the damage
They will screw it up, creating a mutation
crRNA - forms a DNA duplex by binding to the coded section of its 5 end to the matching section of the DNA - the DNA will bind to the RNA rather than its own strand.
The other half of the crRNA is NOT complementary to the DNA strand after the PAM sequence, but the tracrRNA IS complementary to the part of the crRNA which isn’t bound to DNA
This triggers CAS9 to come over and make a double break at the crRNA. The DNA repair enzymes come in to fix it however they don’t know what is missing - makes mistakes/mutations e.g. non-homologous end joining
what DNA can CRISPR cut out?
and what is the base pair for Cas9?
Cas/CRISPR system can cut any piece of genomic DNA that is adjacent to a ‘PAM’ (Protospacer adjacent motif)
These are 2-5 bp sequences that differ between bacterial species
Base pair for for Cas9 is ‘NGG’
How is CAS9 made artificially to work in genes
A section of DNA is added to cells which include the CAS9 gene and crRNA and tracrRNA
What is the difference between CRISPR/CAS in the wild and in the lab
Wild - two separate sections (crRNA and tracrRNA)
Lab - crRNA and tracrRNA fused
Why is CAS9/CRISPR the most popular
Only have to make DNA not proteins
Also affects both alleles not just one
Can do more than one gene at a time
Can inject into zygotes - so a lot quicker than homologous recombination from about 9 months to about 4 weeks
What is one disadvantage of the CAS9/CRISPR system
Mutations induced are uncontrolled and variable
What is homology directered repair (in CRISPR)
Tells the DNA repair enzymes what sequence they have to put in to stop random mutations - can be a whole strand or a single oligonucleotide
Can also add loxP sites for gene knock out
Homology-Directed repair can also be used with TALENs and zinc-finger nucleases
How are animals cloned
Through somatic cell nuclear transfer :
Take a cell from a donor animal –> take the nucleus out of recipient unfertilised oocyte –> put the nucleus of the donor somatic cell into the enucleate oocyte (direct injection/electrofusion) –> activate the oocyte to start development (is reprogrammed by cytoplasm of host to become totipotent again) –> transfer it the the uterus of pseudopregnant female –> completes development as normal in utero
