How does reverse genetics work?
Inactivate the gene and see how development is affected.
Forward genetic screens
Phenotype is generated and the gene responsible is cloned later
CRISPR causes a double stranded break - how is this repaired by the cell? (Two ways)
know this one well
- NHEJ (Non-homologous end joining)
- often results in small insertions or deletions (indel*)
- can cause a premature stop codon - Homology-directed repair
- region of homology inserted via recombination into the double stranded break
How were standard knockouts done before CRISPR!
- Pluripotent cells grown in a culture
- Introduce an altered version of the gene into the cells, let each proliferate to form a colony
- Select targeted cells with a drug (like neomycin)
- Test for the rare colony in which the DNA fragment has replaced one copy of the normal gene
- The cells with one copy of the target get replaced by the mutant gene
- Harvest early embryos from a mouse and inject the mutated ES cells into the isolated embryo
- Hybrid early embryos partly form ES cells
- Introduce the altered embryo into a foster mother
- She gives birth and the babies have a different phenotype
- Now you have a transgenic mouse with one copy of the target gene replaced by altered gene in the germ line
Where are pluripotent stem cells from?
The blastomeres in the inner cell mass
How is a standard gene knockout done?
the neomycin resistance replaces the strand on the gene in order to knock it out, now the organism is resistant to neomycin and can survive on gel
What do you use for conditional gene inactivation
Cre/LoxP
Can delete a gene by flanking with LoxP on either side and it will cut the gene out of the genome
Important Facts About LoxP
- LoxP site is very specific 34bp DNA sequence
- The Cre-recombinase is an enzyme from a bacterial phage that recognizes the LoxP site and deletes the DNA between them
Two components needed for a conditional knockout
- Expression of Cre recombinase in the tissue of interest
2. LoxP sites on either side of the gene that is meant to be deleted
Steps in CRISPR gene editing
- inject homologous DNA with desired sequence along with a plasmid coding for Cas9 and guide RNA
- Make a double stranded break at the editing site
- use homologous recombination or NHEJ
Therapeutic possibilities of CRISPR?
taking human somatic cells (such as fibroblasts) and reprogramming them into induced pluripotent cells (iPS). Here genetic mutations are corrected using CRISPR, then differentiated into the cell type needed and transplanted into the body
What is the biggest concern about using CRISPR
Cutting another site in the genome that wasn’t the desired target
The two ways that scientists are using CRISPR to cure sickle cell anemia
- Fix
- use homologous recombination to fix the adult hemoglobin - Swap
- use fetal hemoglobin instead of the adult one by turning off the BCL11A gene (BCL11A inhibits the production of fetal hemoglobin
CRISPR experiment used to inactivate Rspo2 in frogs (half of them)
inactivated Rspo2 side was similar to a tadpole (wnt signalling pathway)
