1. Genome Editing by Matthew Porteus Department of Pediatrics,
Stanford University School of Medicine,
Stanford, CA, USA;

2. Genome Editing: Definition (2010 Method of the Year, Nature Methods)
Genome editing is the precise modification of the nucleotide sequence of the genome.
A. Genome editing by mutagenic non-homologous end-joining results in nucleotide modification at a precise spatial location in the genome.
B. Genome editing by homologous recombination results in specific and defined changes in the nucleotide content of the genome at a precise spatial location.

3. Two Basic Approaches to Genome Editing
Genome Editing by AAV mediated homologous recombination
Genome Editing by using engineered nucleases to create genome specific DNA double-stranded breaks.

4. Genome Editing Using AAV

5. Four Different Nuclease Platforms to Create Genome Specific Double-Strand Breaks
Homing Endonucleases (HE)
Zinc Finger Nucleases (ZFN)
Tal Effector Nucleases (TALEN)
RNA Guided Endonucleases (RGE, CRISPR/Cas9)

6. Timeline of Development and Use of Engineered Nucleases in Human Cells
2010-Genome Editing “Method of the Year”
1994
2013
1994-Homing Endonuclease (mammalian cell)
1994-Chimeric Nuclease
(in vitro)
1996-Zinc Finger Nuclease
(in vitro)
2003-Zinc Finger Nuclease
(mammalian cell)
2010-Zinc Finger Nuclease
(human clinical trial)
TALEN
2010-non-mammalian cell
2011-mammalian cell
2009-TAL effector code
2012
RGE
(in vitro)
2013
RGE
(mammalian cell)

7. Genome Editing using Engineered Nucleases by Non-Homologous End Joining-I
TALENs
HEs
RGE (CRISPR/Cas9)
ZFNs *
Insertions/Deletions
at Site of Engineered Nuclease Break
(Enhanced by Expression of TREX-2 in certain circumstances)
(To mutate a gene or restore a reading frame)

8. Intra-chromosomal Deletion by Creating Two DSBs on Same Chromosome
Genome Editing using Engineered Nucleases by Non-Homologous End Joining-II
TALENs
HEs
TALENs
HEs
RGE (CRISPR/Cas9)
RGE (CRISPR/Cas9)
ZFNs
ZFNs
Intra-chromosomal Deletion by Creating Two DSBs on Same Chromosome

9. Genome Editing using Engineered Nucleases by Non-Homologous End Joining-III
TALENs
HEs
TALENs
HEs
RGE (CRISPR/Cas9)
RGE (CRISPR/Cas9)
ZFNs
ZFNs
Chromosome A
Chromosome B
Chromosomal Translocation Created by Joining of Two Simultaneous DSBs on Different Chromosomes

10. Genome Editing using Engineered Nucleases by Single Strand Annealing (combination of NHEJ and HR)
TALENs
HEs
RGE (CRISPR/Cas9)
ZFNs
Reduction of Repeat Element by Creating a Break in the Sequence Between the Two Repeats

11. Genome Editing using Engineered Nucleases by Homologous Recombination
TALENs
HEs
RGE (CRISPR/Cas9)
ZFNs
Donor DNA
Donor DNA
Single/Small Defined Nucleotide
Change
Targeted
Transgene(s)
Insertion