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Introduction to Genome Editing
GAO, Bo 高波 Ph.D
School of Biomedical Sciences, Li Ka Shing Faculty of Medicine
The University of Hong Kong
12 July, 2019
YASHK-HKAGE-EDB Talk
orNon-homologous end joining (NHEJ)
Repair mechanisms of double-strand breaks (DSB)
Homology-directed repair (HDR)
Nat Rev Mol Cell Biol. 2013
DSBDSB
Homologous recombination
Spontaneous Homologous Recombination
https://www.intechopen.com/books/cell-interaction/
Homologous armHomologous arm
Spontaneous homologous recombination is rare in normal cells
Nuclease-mediated DSB to enhance homologous recombination
Enhancement of Homologous Recombination by creating DSB
https://www.intechopen.com/books/cell-interaction/
I-SceInuclease
Enhancement of Homologous Recombination by creating DSB
Engineered nuclease to make DSB at specific site
https://www.intechopen.com/books/cell-interaction/
DNA-binding domain
DNA-cutting domain
Zinc finger nuclease (ZFN)
Transcription activator-like effector nuclease (TALEN)
Nat Rev Genet. 2013
Module 1 Module 2
Bacteria vs Viruses: The Biggest Tiniest War
Virus for bacterium – bacteriophage, also known as phage
What is CRISPR ?
In 1987 Ishino et al. first described a pattern of shortpalindromic repeats of DNA interspaced with short, nonrepetitive “spacers” of DNA in E.coli bacteria
http://crispr.i2bc.paris-saclay.fr
J. Bacteriol. 1987
Direct Repeat
Reverse
(Palindromic sequence)
Direct Repeat Spacer
With time more such patterns were observed in other bacteria andarchaea and in 2002, Jansen et al. named the pattern CRISPR, short for“clustered regularly interspaced short palindromic repeats” and alsodocumented the existence of a number of CRISPR-associated genes(named the Cas family) adjacent to these repeats.
What is CRISPR ?
Mol. Microbiol. 2002
Cell, 2014Cas genes: CRISPR-associated genes
In 2007, scientists showed that CRISPR provides bacterial immunityagainst viruses by matching DNA in spacer sequences with DNA from virus
There is CRISPR in Your Yogurt !
The research was carried by scientists in , a food company
matches
Virus
Science, 2007
non-virus-resistant
bacteria
Extra spacer
virus-resistant
bacteria
http://doudnalab.org/
Genome editing in test tube
Cas Guide RNA
Target DNA
Cas:RNA complex
Target DNA
Guide RNA
Genome editing in human cells
Science, 2013
DSBDSB (double strand break)
Homologous recombination
Non-homologous end joining (NHEJ) Homology-directed repair (HDR)
NHEJ-mediated random indel mutation HDR-mediated precise knock-in
Genome editing in mouse embryos
Targeting Vector
Construction
Cell, 2013 May
Cas
Guide RNA
Donor template
The Heroes of CRISPR
David Liu
Harvard
Base editing
Philippe Horvath
Danisco / Dupont
Jennifer Doudna
UC BerkeleyEmmanuelle Charpentier
Max Planck Institute
Feng ZhangMIT
George Church
Harvard
CRISPR as a bacterial immune system Developed CRISPR-mediated genome editing
Application in mammalian cells
Application in animal models