Genome Editing Talk, Veterinary Pathology Rounds, UC Davis, 20160617

1. CRISPR-Cas9
Genome Editing
20160617
Lab
Animal
Pathology
Rounds
Han
Tan
(ekhtan@ucdavis.edu)
TwiCer:
@ekhtn

2. Why Genome Editing?
§ Accelerate
basic
research
§ Generate
mulGple
alleles
§ Generate
mulGple
gene
mutaGons
§ Analysis
of
linked
genes
§ Analysis
of
lethal
genes
§ Disease
modeling
§ Gene
Therapy
§ Replace
defecGve
genes
§ Fix
specific
cell-­‐types
§ Agriculture
§ Non-­‐transgenic
approaches
to
improve
crops
§ Genome
engineering
of
plants
and
animals
§ Biotechnology
§ Ecological
control
of
vectors
that
transmit
diseases
§ SyntheGc
biology

3. Engineering POLLED
Phenotype in Dairy Cows
ele
e
Figure 1 Phenotypic and genotypic confirmation of POLLED introgression in Spotigy and Buri. (a,b)
Diagnostic PCRs for the Pc allele using primer pairs btHP-F1 + btHP-R2 (a) and HP1748-F1 +
HP1594_ 1748-R1 (b) (Supplementary Methods), respectively, confirmed homozygous introgression in
RCI-001, Spotigy (RCI-002) and Buri (RCI-003), and heterozygous introgression in RCI-004 relative to
the donor cell line that is negative. The identity of PCR products was confirmed by Sanger sequencing.
The positive control (p1748) was a plasmid containing the Pc allele10. (c) Photograph of Spotigy at
2 months of age, so named after the black spots where horn buds would have developed. (d)
900 3,000
2,500
2,000
1,500
1,000
700
600
500
400
300
001 002 003 004 2120p1748 001 002 003 004 t2120p1748
c d
(Carlson
et
al.,
Nature
Biotech.
2016)
*Instant
introgression
directly
into
elite
breeds
–
bypasses
tradiGonal
breeding

4. Outline
§ Genome
EdiGng
§ The
CRISPR-­‐Cas9
System
§ The
many
flavors
of
Cas9
§ Genome
EdiGng
in
the
lab
and
beyond

5. What is Genome Editing?
§ Targeted
modificaGon
to
DNA
How?
§ Using
customized
sequence-­‐specific
nucleases

6. Zinc Finger Nucleases
(ZFNs)
(Baltes
et
al.,
Trends
in
Biotech.
2015)

7. Transcription Activator-Like
Effector Nucleases (TALENs)
(Baltes
et
al.,
Trends
in
Biotech.
2015)
N-­‐
-­‐C
Repeat
Variable
Diresidue

8. Cas9
CRISPR-Cas9
sgRNA
NGG
NGG

9. Comparisons
ZFNs
TALENs
CRISPR-­‐Cas9
DNA
recogni=on
MulGmeric
protein
–
DNA
interacGon
Protein
–
DNA
interacGon
RNA
–
DNA
Watson-­‐
Crick
base-­‐pairing
DNA
cleavage
Coupling
to
non-­‐
specific
nuclease
FokI
Coupling
to
non-­‐
specific
nuclease
FokI
Innate
to
Cas9
Requirements
Two
large
protein
constructs
Two
large
protein
constructs
Simple
20nt
change
to
construct
Targe=ng
Poor
Good
Good
Feasibility
Difficult
Difficult
Easy

10. CRISPR vs TALEN
as
of
6/16/16

11. Outline
§ Genome
EdiGng
§ The
CRISPR-­‐Cas9
System
§ The
many
flavors
of
Cas9
§ CRISPR-­‐Cas9
in
the
lab
and
beyond

12. Discovery of CRISPR-Cas
§ CRISPR
=
Clustered
Regularly
Interspersed
Short
Palindromic
Repeats
[DNA
repeats]
§ Cas
=
CRISPR
associated
[Protein
coding
sequences]
§ Discovered
in
1987
from
the
analysis
of
E.
coli
genomes
(Ishino
et
al.,
J.
Bacteriol.
1987)
§ Is
important
for
adapGve
immunity
in
bacteria
and
archaea

13. Type II CRISPR-Cas
which require substantial protein engineering for
each DNA target site to be modified, the CRISPR-
Cas9 system requires only a change in the guide
RNA sequence. For this reason, the CRISPR-Cas9
protein Cas9 together with suitable sgRNAs. As
discussed above, CRISPR refers to the repetitive
nature of the repeats in the CRISPR arrays that
encode crRNAs, and the term does not relate
the crRNA repeat–
tion (77, 78). An
bridges the two str
pears to be the hin
(Doudna
&
CharpenGer,
Science
2014)
strand invasion and RNA-DNA hybrid forma-
tion (80–82).
To assess the target-binding behavior of Cas9
in cells, researchers used chromatin immuno-
precipitation and high-throughput sequencing
(ChIP-seq) to determine the numbers and types
of Cas9 binding sites on the chromosome. Re-
sults showed that in both human embryonic kid-
ney (HEK293) cells (83) and mouse embryonic
stem cells (mESCs) (84), a catalytically inactive
version of Cas9 bound to many more sites than
those matching the sequence of the sgRNA used
in each case. Such off-target interactions with
DNA, typically at sites bearing a PAM and par-
tially complementary to the guide RNA se-
quence, are consistent with established modes
as in mouse cells (75). The expected alterations
in the target DNA were observed, indicating
that site-specific DSBs by RNA-guided Cas9 had
stimulated gene editing by nonhomologous end
joining repair or gene replacement by homology-
directed repair (Fig. 4). Targeting with multiple
sgRNAs—referred to as multiplexing—was also
successfully achieved (75, 86). RNA-programmable
S. pyogenes Cas9-mediated editing has now been
applied to various human cells and embryonic
stem cells [(87–90); for reviews, see (91–93)]. Al-
though direct comparisons can be difficult to
assess because of differences in target sites and
protein expression levels, some analyses show
that CRISPR-Cas9–mediated editing efficiencies
can reach 80% or more depending on the target,
gene functio
scale lentivi
to generate
screening a
and negative
was also us
cell viability
(102). Althou
using RNA
expression o
the generati
fer from sub
of CRISPR-
enable large-
other phenot
and utility of
Streptococcus
pyogenes
Protospacer
Adjacent
MoGf
CRISPR
RNA
Trans-­‐acGvaGng
CRISPR
RNA

14. CRISPR-Cas9
M and par-
e RNA se-
hed modes
protein expression levels, some analyses show
that CRISPR-Cas9–mediated editing efficiencies
can reach 80% or more depending on the target,
enable large-scale screening
other phenotypes and thus w
and utility of genetic screen
nonmodel cel
Other per
CRISPR-Cas9
evance to hu
the ability to
tions respons
orders. A do
the Crygc g
cataracts was
in mice (103)
mary adult
derived from
the CFTR lo
cystic fibros
homologous r
ing in the c
miniature or
(organoids) h
exact genetic
as9. The structure of S. pyogenes Cas9 in the unliganded and RNA-DNA–bound
sgRNA
(single
guide
RNA)
(Jinek
et
al.,
Science
2012
Doudna
&
CharpenGer,
Science
2014)

15. What
can
you
do
with
double-­‐stranded
DNA
breaks?

16. DNA Repair by Non-Homologous
End-Joining (NHEJ)
DNA
LIGASE
IV
(LIG4)
XRCC4
KU70/KU80
Untemplated
DNA
repair
and
very
error
prone
–
deleGons,
inserGons,
rearrangements
etc.

17. Usefulness of DNA Repair by NHEJ
§ An
effecGve
form
of
mutagenesis
§ Diversity
of
breakpoints
repaired
by
NHEJ
=
mulGple
alleles
are
generated
instantly
§ Make
two
breaks
for
large
deleGons
§ Make
mulGple
breaks
for
translocaGons,
desired
rearrangements,
cut
and
paste
modificaGons

18. Using a sgRNA Library to Screen
Mammalian Cells
cin, len
in 93 T
(Fig. 1
reveale
sequen
contras
vectors
to inco
(Fig. 1C
Giv
by lent
ducting
(GeCK
library.
ing 5′ c
in the h
of 3 to
target s
ificatio
To t
at achi
gets, w
profilin
al deliv-
sgRNA
ntdeple-
nes. (A)
sion vec-
d sgRNA
ro, puro-
marker;
ngsignal;
element;
urinetract;
actor-1a
P2A, 2A
de;WPRE,
nal regu-
B) Distri-
nce from
ansduced
ng lenti-
1 to 6,
nd Cas9-
ed peak)
uorescent 293T cells (gray shaded peak). (C) Distribution of fluorescence from 293T-EGFP cells
A
B C
sgRNAU6 EFS SpCas9 WPREP2A Puro
cPPT
RREpsi+
lentiCRISPR
EGFP fluorescence (a.u.)
Normalizedcellcount(a.u.)
Normalizedcellcount(a.u.)
100
80
60
40
20
0
10
0 2 4 6
10 10 10
Cas9 only
HEK293T
sgRNA 1
sgRNA 2
sgRNA 3
sgRNA 4
sgRNA 5
sgRNA 6
Cas9:sgRNA
100
80
60
40
20
0
10
0 2 4 6
10 10 10
control shRNA
HEK293T
shRNA 1
shRNA 2
shRNA 3
shRNA 4
EGFP fluorescence (a.u.)
shRNA
(Shalem
et
al.,
Science
2014)

19. A B
D
Step1:
sgRNAoligo
librarydesign
Step2:
Constructionof
lentiCRISPRscreening
library
Oligo array
synthesis
lentiCRISPR
library
Cloning sgRNA
oligo libray
into lentiviral
constructs
Step3:
GeCKO
Transduction
with lentiCRISPR
library
Select for
transduced cells
and apply
screening
assay
Analysis
of remaining
sgRNA pool
A375
Day 14
Day 3
Log2 normalized gene
Cumulativefrequency
0
0.2
0.4
0.6
0.8
1
2 4 6 8
RNA processing
Structural constituent
of ribosome
Ribonucleoprotein
complex (RNPC)
RNPC biogenesis
and assembly
RNA binding
Gene ran
2000 6000 10000
Depleted E
(Shalem
et
al.,
Science
2014)

20. DNA Repair by Homology-
Directed Repair (HDR)
RAD51
Templated
DNA
repair
using
homologous
sequences
D-­‐Loop
formaGon
and
DNA
synthesis

21. Usefulness of DNA Repair by HDR
§ Gene
Therapy
§ Gene
Replacement
§ Genome
Engineering
§ SyntheGc
Biology
§ Gene
Drive
–
Control
vectors
such
as
mosquitoes

22. Correcting a Gene
(Mouse Model)
• FAH
gene
–
last
gene
in
the
tyrosine
catabolic
pathway
• Recessive
mutaGon
of
GàA
in
exon
8
causes
Hereditary
Tyrosinemia
and
is
fatal
using the mismatch-spe
detected at Fah in 3T3
match between FAH1,
prevent Cas9-mediated
was not detected at the a
xon 8 and Fahmut/mut mice injected with FAH1, 2
- and 405-bp PCR bands, indicating that the exon
is restored in a subset of hepatocytes (Fig. 2b).
05-bp bands in CRISPR-Cas9 treated mice con-
cted G nucleotide is included in the PCR product
Hydrodynamic injection
(Cas9 + sgRNA + ssDNA)
NTBC withdrawn
Fahmut/mut Fah+ hepatocytes?
5…CCTCATGAACGACTGGAGCGgtaatgcctggtgg…3 ssDNA
5…CCTCATGAACGACTGGAGCAgtaatgcctggtgg…3 genomic
7 8 9 7 8 9
A G
a
d
Fah+/
H&E
a
Fah IHC
Fahmut/mut
Unguided Cas9Fah+/+
FAH2
NTBC off NTBC off
Figure 2 CRISPR-Cas9–mediated editing corrects Fah s
the liver. (a) Fah immunohistochemistry (IHC) of Fahmut/m
with unguided Cas9 or Cas9 plus the FAH2 sgRNA. Uppe
were off NTBC water for 30 d as in Figure 1d. There are 3
Fah+ cells (n = 3 mice). Lower panel: mice were kept on
euthanized at day 6 to estimate initial repair rate. Fah+ c
0.40 0.12% for FAH2 and 0.01 0.02% for unguided
(n = 3 mice) using an unpaired t-test. Fah+/+ mice are sh
Scale bars, 100 m. (b) RT-PCR in liver RNA from wild-t
Fahmut/mut and Fahmut/mut mice injected with FAH1, 2 or
B R I E F C O M M U N
(Lin
et
al.,
Nature
Biotech.
2014)
prevent Ca
was not de
Sequencing of the 405-bp bands in CRISPR-Cas9 treated mice con-
firmed that the corrected G nucleotide is included in the PCR product
Figure 1 Hydrodynamic
injection of CRISPR
components rescues
lethal phenotype of
Fah-deficient mice.
(a) Experimental
design. Fahmut/mut
mice harbor a
homozygous G A
point mutation at
the last nucleotide of
exon 8 (red), causing
skipping of exon 8
during splicing. pX330
plasmids expressing
Cas9 and a sgRNA
targeting the Fah locus
are delivered to the
liver by hydrodynamic
tail vein injection. A
ssDNA oligo with the
correct fragment of
Fah sequence (i.e., the
G allele) is co-injected
Hydrodynamic injection
(Cas9 + sgRNA + ssDNA)
NTBC withdrawn
Fahmut/mut Fah+ hepatocytes?
5…CCTCATGAACGACTGGAGCGgtaatgcctggtgg…3 ssDNA
5…CCTCATGAACGACTGGAGCAgtaatgcctggtgg…3 genomic
7 8 9 7 8 9
A G
a
b
d
1.1
Injection
day
(–3)
7
8
11
13
14
15
16
20
21
22
23
24
25
26
27
28
29
30
Day 0
Time (days)
Weightratio
FAH1
FAH2
FAH1
FAH3
Saline
ssDNA oligo
Unguided Cas9
0.9
0.8

23. Outline
§ Genome
EdiGng
§ The
CRISPR-­‐Cas9
System
§ The
many
flavors
of
Cas9
§ Genome
EdiGng
in
the
lab
and
beyond

24. Various Flavors of Cas9
cellular DNA repair
hat catalyze non-
end joining (NHEJ)
-directed repair
as9 can function as
Cas9) when engi-
ntain an inactivat-
in either the HNH
uvC domain active
nCas9 is used with
that recognize
sites in DNA, a
ouble-strand break
C) Cas9 functions
uided DNA binding
n engineered to
ivating mutations
active sites. This
nactive or dead
9) can mediate
al down-regulation
, particularly
to activator or
mains. In addition,
e fused to fluores-
s, such as green
protein (GFP), for
ging of chromo-
ther dCas9 fusions,
e including chro-
A modification
y enable targeted
hanges to
A.
onMay20,2016http://science.sciencemag.org/Downloadedfrom
onMay20,http://science.sciencemag.org/Downloadedfrom
§ Higher
specificity
and
efficiency
than
using
a
one
sgRNA
§ Similar
to
TALENs
Single-­‐strand
DNA
breaks
are
repaired
slightly
differently
than
dsDNA
breaks
(Doudna
&
CharpenGer,
Science
2014)

25. 28 NOVEMBER 2014 • VOL 346 ISSUE 62
ns,
d
§ Repression
of
genes
§ AcGvaGon
of
genes
§ Fluorescent
tags
§ EpigeneGc
modificaGons
§ DNA
pull-­‐downs
§ dCas9
can
even
be
used
for
HDR
dCas9
=
“dead”
Cas9
(Doudna
&
CharpenGer,
Science
2014)

26. Outline
§ Genome
EdiGng
§ The
CRISPR-­‐Cas9
System
§ The
many
flavors
of
Cas9
§ Genome
EdiGng
in
the
lab
and
beyond

27. Cpf1
(Zetsche
et
al.,
Cell
2015)
Another
Type
II
CRISPR-­‐Cas
system

28. NgAgo
P
Phosphorylated
ssDNA
as
the
guide
(Gao
et
al.,
Nature
Biotech.
2016)
Argonaute
from
Natronobacterium
gregoryi
NgAgo
dsDNA
target

29. C2c2 CRISPR for RNA
targeting
(Abudayyeh
et
al.,
Science
2016)
C2c2
crRNA
RNA
Class
II
Type
VIA
CRISPR-­‐Cas
system
from
Leptotrichia
shahii

30. Take Home
§ Genome
ediGng
is
a
powerful
tool
that
is
under
constant
development
–
CRISPR-­‐Cas
systems
in
parGcular
§ The
advantages
of
CRISPR-­‐Cas9
are
its
ease
of
use
and
its
efficacy
in
almost
any
organism
that
has
been
tested
so
far
§ Roadblock
to
genome
engineering
has
been
removed
with
CRISPR-­‐Cas9
technology
§ The
potenGal
impacts
that
genome
engineering
can
have
in
our
lives
is
just
emerging
–
stay
tuned!