BCH3031 Lecture Notes - Lecture 20: Zinc Finger Nuclease, Dna-Binding Domain, Zinc Finger
25 May 2018
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Lecture 20
How do you modify gene function in vivo?
• Desirable because want to find out what a gene does – deletion
o Generally
o At a particular time
▪ Developmental vs. adult phase
o In a particular place
▪ After birth, in adulthood
• Knockout/modification by homologous
recombination in ES cells (reverse genetics)
o Isolate blastocyst and inner cell mass →
put into cultured ES cells → contribute
to every cell in body → inject into
blastocyst → take embryos and put in
mouse → chimaera mouse cross with G2
o Selecting for white cells into germline
How else can we Modify the Genome of an Organism?
• TALENs – transcription factor like effector
nucleases
o Derived from bacteria
o DNA binding domain contains repeated
highly conserve 33-34 amino acid
sequences
▪ Strong correlation with specific nucleotide recognition
• ZFNs – zinc finger nucleases
o Artificial restriction enzymes generated by fusing zinc finger DNA-
binding domain to a DNA-cleavage domain
o Zinc finger domains can target unique sequences within complex
genomes
o Limitations: sequence specificity, limited to where you can target
genome
• Problem with TALENs and ZFNs
o Assembling fingers and TALEs – bit of a faff
o Costs for commercial delivery can be very high
o Immunogenicity
o Specificity
o Efficiency
• CRISPR-Cas9
o 1987: found oddly repeated sequence at end of bacterial gene
o 1990s: found 40% of bacteria have them, 90% of archaea have them
o 2005: space DNAs s
o 2007: if you change spacer DNA sequences → affects resistance to
phage
o 2011: Cas9 + tracrRNA + transcribed crispr locus = DNA specific
nuclease
o 2012: Can make synthetic tracr and guide RNA hybrid will cut DNA at
specific sites upon addition of Cas9 protein – custom genome editing
o Phase 1: immunization
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