1. The beginnings of gene editing
Maria Jasin, PhD
Memorial Sloan Kettering Cancer Center
New York
Courtesy of J Doudna
(H. Adam Steinberg, artist)

2. Understanding biological characteristics
Phenotype
Forward genetics
Genotype

3. Scientists: to understanding biological characteristics
Why modify the genome?
Scientists: to understanding biological characteristics
Forward genetics
Genotype
Reverse genetics
Phenotype

4. Physicians: to ameliorate human disease
Why modify the genome?
Physicians: to ameliorate human disease
Sharon Lees/ Great Ormond Street Hospital
Reverse genetics
Acute lymphoblastic leukaemia (ALL) is the most common type of cancer affecting children.
Around one in every 2,000 children will develop ALL, with most cases occurring between the ages of two and five.
Symptoms of ALL usually begin slowly before rapidly getting severe.
They include pale skin, tiredness, breathlessness, having repeated infections over a short space of time and unusual and frequent bleeding.
Usually, the bone marrow in the body produces stem cells which are allowed to fully develop into other cells before being released into the blood.
But in ALL, the bone marrow starts releasing large numbers of immature white blood cells. These are known as blast cells.
As they increase, there is a corresponding drop in the number of red blood cells and platelet cells, causing symptoms of anaemia.
Read more: Follow on Twitter | DailyMail on Facebook Sharon Lees/ Great Ormond Street Hospital
NIH.gov/science/hiv

5. Targeted genome modification at the end of the 20th century
neoR
Homologous integration
βS-globin βS
βA-globin
1 in 9700 clones
neoR βA
Oliver Smithies and colleagues, 1985, 1991

6. Targeted genome modification at the end of the 20th century
neoR
Homologous integration
βS-globin βS
βA-globin
0.01%
neoR βA
Random integration
99.99%
neoR
Oliver Smithies and colleagues, 1985, 1991

7. Targeted genome modification at the end of the 20th century
neoR
Homologous integration
βS-globin βS
βA-globin
0.01%*
neoR βA
Random integration
99.99%
neoR
*~1 in 106 in the total population of total cells
Oliver Smithies and colleagues, 1985, 1991

8. Targeted genome modification at the end of the 20th century
Highly inefficient: ~1 in 106 unselected cells; ~1 in selected cells
Possible only in cell lines that can undergo selection, including mouse embryonic stem cells
Thus, not applicable to most other organisms than the mouse, including humans
The exception is yeast…

9. Yeast: Targeted genome modification occurs in 100% of selected cells
Homology
Homologous integration
Chromosome
Plasmid
Chromosome
Figure 1. DSB-induced homologous recombination (HR) between chromosomal and plasmid DNA. Homology between the chromosome and plasmid is represented by the red bar.
A. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by a simple gene conversion. Such noncrossovers can be detected if the plasmid contains an origin of replication.
B. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by gene conversion with a crossover, leading to plasmid integration during gene targeting. Crossovers are suppressed in mammalian cells.
C. A DSB in the chromosome can be repaired using the plasmid as a template, to resulting in efficient genome modification.

10. DNA double-strand break (DSB)

11. Yeast: DSB in the plasmid makes an already highly feasible outcome more efficient
Homologous integration
Chromosome
DSB
Plasmid
~100-fold
more efficient
Chromosome
Figure 1. DSB-induced homologous recombination (HR) between chromosomal and plasmid DNA. Homology between the chromosome and plasmid is represented by the red bar.
A. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by a simple gene conversion. Such noncrossovers can be detected if the plasmid contains an origin of replication.
B. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by gene conversion with a crossover, leading to plasmid integration during gene targeting. Crossovers are suppressed in mammalian cells.
C. A DSB in the chromosome can be repaired using the plasmid as a template, to resulting in efficient genome modification.

12. Mammalian cells: DSB in the plasmid also increases homologous integration
Chromosome
DSB
Plasmid
~100-fold
more efficient
Chromosome
Figure 1. DSB-induced homologous recombination (HR) between chromosomal and plasmid DNA. Homology between the chromosome and plasmid is represented by the red bar.
A. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by a simple gene conversion. Such noncrossovers can be detected if the plasmid contains an origin of replication.
B. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by gene conversion with a crossover, leading to plasmid integration during gene targeting. Crossovers are suppressed in mammalian cells.
C. A DSB in the chromosome can be repaired using the plasmid as a template, to resulting in efficient genome modification.
High frequency of homologous recombination in mammalian cells.
M Jasin, J de Villiers, F Weber, W Schaffner
Cell 1985 Dec;43:
Homologous integration in mammalian cells without target gene selection.
M Jasin, P Berg.
Genes Dev Nov;2(11):

13. Mammalian cells: DSB in the plasmid also increases homologous integration
Chromosome
DSB
Plasmid
but still frequent
random integration
Chromosome
Figure 1. DSB-induced homologous recombination (HR) between chromosomal and plasmid DNA. Homology between the chromosome and plasmid is represented by the red bar.
A. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by a simple gene conversion. Such noncrossovers can be detected if the plasmid contains an origin of replication.
B. A DSB or gap in plasmid DNA can be repaired using the chromosome as a template by gene conversion with a crossover, leading to plasmid integration during gene targeting. Crossovers are suppressed in mammalian cells.
C. A DSB in the chromosome can be repaired using the plasmid as a template, to resulting in efficient genome modification.
Some basic principles from yeast hold in other organisms, but others do not

14. Yeast: Double-strand break model for recombination
DSB or
DSB repaired from
homologous sequence
Jack W. Szostak, Terry L. Orr-Weaver,
Rodney J. Rothstein, and Franklin W. Stahl
Cell, vol May 1983

15. In 1994, our basic premise for targeted modification of the genome:
Introduce a DSB into the chromosome, rather than plasmid;
Cellular DNA repair mechanisms will repair the DSB, modifying the chromosome

16. In 1994, our basic premise for targeted modification of the genome:
Introduce a DSB into the chromosome, rather than plasmid;
Cellular DNA repair mechanisms will repair the DSB, modifying the chromosome

17. First gene editing experiment
DSB:
I-SceI endonuclease
(a meganuclease)
I-SceI site
Chromosome
Figure 2. Gene editing induced by a DSB in the chromosome. A DSB in the chromosome can be repaired by recombination with introduced homologous DNA, i.e., gene targeting, (A) or by imprecise NHEJ leading to mutagenesis (B). Initial studies in mammalian cells with I-SceI endonuclease showed that both kinds of events were efficiently induced, with imprecise NHEJ events somewhat more abundant. Experiments with ZFNs,TALENS, and CRISPR/Cas9 have also shown that both types of gene editing are induced by these nucleases as well.
Rouet et al, 1994

18. First gene editing experiment
DSB:
I-SceI endonuclease
(a meganuclease)
I-SceI site
Chromosome
Figure 2. Gene editing induced by a DSB in the chromosome. A DSB in the chromosome can be repaired by recombination with introduced homologous DNA, i.e., gene targeting, (A) or by imprecise NHEJ leading to mutagenesis (B). Initial studies in mammalian cells with I-SceI endonuclease showed that both kinds of events were efficiently induced, with imprecise NHEJ events somewhat more abundant. Experiments with ZFNs,TALENS, and CRISPR/Cas9 have also shown that both types of gene editing are induced by these nucleases as well.
Rouet et al, 1994

19. First gene editing experiment
DSB:
I-SceI endonuclease
(a meganuclease)
I-SceI site
Chromosome
Homologous recombination
Chromosome
Gene targeting
Figure 2. Gene editing induced by a DSB in the chromosome. A DSB in the chromosome can be repaired by recombination with introduced homologous DNA, i.e., gene targeting, (A) or by imprecise NHEJ leading to mutagenesis (B). Initial studies in mammalian cells with I-SceI endonuclease showed that both kinds of events were efficiently induced, with imprecise NHEJ events somewhat more abundant. Experiments with ZFNs,TALENS, and CRISPR/Cas9 have also shown that both types of gene editing are induced by these nucleases as well.
Rouet et al, 1994

20. First gene editing experiment
DSB
Homologous recombination (HR)
Gene targeting
Nonhomologous
end-joining (NHEJ)
Mutagenesis
(indels) //
Figure 2. Gene editing induced by a DSB in the chromosome. A DSB in the chromosome can be repaired by recombination with introduced homologous DNA, i.e., gene targeting, (A) or by imprecise NHEJ leading to mutagenesis (B). Initial studies in mammalian cells with I-SceI endonuclease showed that both kinds of events were efficiently induced, with imprecise NHEJ events somewhat more abundant. Experiments with ZFNs,TALENS, and CRISPR/Cas9 have also shown that both types of gene editing are induced by these nucleases as well.
Rouet et al, 1994

21. Gene editing: DSB-induced genome modification
Two outcomes:
targeted modification by HR or mutagenesis by NHEJ
NHEJ known for its use generating diversity in immune system rearrangements
Highly efficient
In principle, possible in any cell or organism
Time after I-SceI expression hr
NHEJ HR
Liang et al 1998

22. endonuclease/cleavage site
I-SceI
endonuclease/cleavage site
DSB
Homologous recombination (HR)
Nonhomologous
end-joining (NHEJ) //
Figure 2. Gene editing induced by a DSB in the chromosome. A DSB in the chromosome can be repaired by recombination with introduced homologous DNA, i.e., gene targeting, (A) or by imprecise NHEJ leading to mutagenesis (B). Initial studies in mammalian cells with I-SceI endonuclease showed that both kinds of events were efficiently induced, with imprecise NHEJ events somewhat more abundant. Experiments with ZFNs,TALENS, and CRISPR/Cas9 have also shown that both types of gene editing are induced by these nucleases as well.
Problem for protein engineering:
Multiple contacts with DNA across the 18bp site

23. DSBs at any genomic site using designed endonucleases
Zn Finger nucleases
(ZFN)
FokI
TALE nucleases
(TALENs)
RNA directed cleavage:
Cas9/guide RNA
ZnF
TAL
Cas9
FokI
guide
RNA
Code for DNA recognition:
~3 amino acids to 3 bp ~1 amino acid to 1 bp Watson-Crick bp
Gene modification:
Modify some genes
(2005)
Modify any gene
(2009)
Multiple genes
(2012)

24. DSBs induce chromosomal rearrangements in mammalian cells
Parental cell line
Reciprocal translocation
Richardson and Jasin, Nature 2000
Also ZFNs, TALENs, Cas9, pnCas9 24

25. Gene editing: DSB-induced genome modification
Two outcomes:
targeted modification by HR or mutagenesis by NHEJ
Highly efficient
In principle, possible in any cell or organism
Danger of unintended consequences (e.g., genetic rearrangements)