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Genome Editing - Coggle Diagram
Genome Editing
Programmable Nucleases
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induce site-specific DNA double strand breaks which enhance the efficiency of homologous recombinaiton and / or trigger error-prone NHEJ which leads to target mutagenesis
shared features :
⁌ Repair of DNA double stranded breaks
⁌ Gene disruption
⁌ Gene Insertion
⁌ Gene correction and point mutation
⁌ Chromosomal rearrangement
ZINC FINGER NUCLEASES
structure ⇒ 2 domains:
DNA-binidng zinc-finger protein (ZFP) domain
nuclease domain derived from the Fokl restriction enzyme
two Zinc-finger monomers are required to form an active nuclease → this requirement for dimerization doubles the lenght of the recognition sites that increses the specificity
the sequence specificity is determined by ZFPs
☛ they recognize a 3-bp DNA sequence.
☞ DNA-bining specificities of zinc-fingers can be altered by mutagenesis
There are no open-source collection of 64 zinc-fingers that cover all possible combinations of triplet sites
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CRISPR-Cas9
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➳ capture small DNA fragments (~ 20bp) from the forein DNA of invading phages or plasmids and insert these sequennes into their own genome to form a CRISPR
Type II CRISPR ➔ these regions are transcribed as pre-CRISPR RNA (pre-crRNA) and processed to give target specific crRNA
trans-activating crRNA (tracrRNA) is also transcribed from the locus and contributes to the processing for pre-crDNA
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Advantages →
▸ RGNEs are simple to design and prepare
▸ new RGENs can be made without clonign
▸ Can cleave methylated DNA
Disadvantages →
▸ Cas9 requires the PAM sequence (not all sequences that contina the PAM sequence are cleved efficiently)
Cas9
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we can manipulate the Cas9 and delete the endonuclease domain to link an activator domain → it becomes a gene activator (o rwe can also do a repressor and regulator)
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