ANHB3323 Lecture Notes - Lecture 6: Apoptosis, Crispr, Telomerase
3 Jun 2018
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LECTURE SIX: Genome Engineering
Genome Editing:
• To induce changes to the sequence of genomic DNA in a controllable
manner
• Occurs in living cells in an efficient way that is permanent
• Editing genomic content in vivo
• Done without any other unwanted modifications
Applications of Genome Engineering:
• Change something → creating/correcting genetic mutation
• Add something → tagging a gene with a reporter gene
• Remove something → delete a gene out of the genome (KO)
DNA Breaks and Repair Mechanisms:
• 1st step in genome engineering → introducing break in DNA in desired
part of genome
• Single strand break → nick → more common, many mechanisms to fix
• Double strand break → cut → rare, catastrophic if happens
• Caused by
o Radiation
o Reactive oxidative stress
o Other chemical attacks
• Non-homologous end joining (NHEJ)
o Fixing cuts
o Error prone
o Faster method
o More frequently used
o Ends of DNA glued back together → no sequence homology
involved but some micro-homology can occur
o Can result in deletions or insertions indels
• Homology directed recombination (HDR)
o Fixing cuts and nicks
o High fidelity
o Slower as it needs to find repair template
o Relies on having another piece of intact DNA to use as a template
to fix broken part → usually other chromosome
o Machinery excises DNA to find perfect matches and fills in using
sister chromatin as template
• Both NHEJ and HDR are naturally occurring processes in the cell
Genome Editing Technologies:
• Induce guided DNA cut or nick → using NHEJ or HDR and exogenous DNA
repair template to form new DNA sequence
• Technologies
o Designer zinc finger proteins → ZFs → transcription factors →
early 2000s
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