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

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.

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.

Genome Editing Using AAV

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)

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)

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)

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

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

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

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