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

http://www.genosense.com

DNA is the code of life

Genome Editing

DNA Damage

https://www.sierraoncology.com

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

ZFN TALEN

Science, 2014

CRISPR

Popular genome-editing tools using nucleases

The rise of CRISPR

Nature, 2015

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

CRISPR is a bacterial immune system

CRISPR: From Yogurt to Genome Editing

Cell, 2014

http://doudnalab.org/

Genome editing in test tube

Cas Guide RNA

Target DNA

Cas:RNA complex

Target DNA

Guide RNA

CRISPR-Cas9: From Yogurt to Genome Editing

Cell, 2014

Feng Zhang, MIT

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

Development, 2014

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