Summary
summary of the crisprcas lecture 2024
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summary of the crisprcas lecture 2024
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GENOME EDITINGGenome editing makes targeted changes in the DNA of a living cell or organism.
It can be used to add or remove DNA in the genome or to alter its sequence.
A. Tagging of “protein of interest” with e.g. GFP, venus
a. Can tag a protein to visualize it under the microscope.
i. Fluorescent tag
B. To create mutant mice/cells to study a disease or
biological process
a. By adding a mutation that is found in
humans and add it to another cell to make
it mutant.
C. To remove mutations in patients to cure a disease
Q1: you want to study the effects of a mutation by comparing the behavior of cells
expressing normal and mutant proteins genome editing is always preferred over
transfection/transduction of WT mutant over expression constructs
TRUE: for the biological POV: not truncated to express large volumes able to get
artefacts because the cell is made to fold normal levels not excessive cellular
phenotype is not attributed to the … as such.
o We assume that the transfection efficiency will be the same
Expressed at physiological levels and time points
- No artefacts attributed to excessive expression levels
- WT/mutant should express similarly (protein of interest)
- No confounding effect of endogens
FALSE: for the technical POV: suboptimal sensitivity of tools for subsequent
functional analyses
o Some Ab cannot detect low protein levels
o Lack of Ab specifity in some cases
HOW IT ALL STARTED
2 repair mechanisms
- Add a repair template is its available insert point mutation in cells
- Non homologous end joining breaks will be ligated again BUT error prone
o Can result in a knock out or LOF
, Only an ending number of nucleases we can utilize can’t just make all the specific
point mutations
NEED for alternatives
ALTERNATIVES
ZNF: zinc finger nucleases
- Artificial endonucleases
- Custom DNA target
Consists of 2 parts
1. DNA-binding domains of transcription factors linked to nuclease domain of Fok1
(protein arm)
Modular zinc finger protein, each module recognizes ±3 bp (up to 6 modules)
2. Two arms – Fok1 functions as a dimer
Disadvantages: time consuming and not that straight forward, need for a specific zinc
finger
NEED 2 arms for active cutting
Concluding: better but not optimal
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