Cas9-mediated genome editing in hematopoietic stem/progenitor cells
J.L. Gori, G.G. Welstead, J. M. Heath, M.A. Collins, J.W. Fang, A.E. Friedland, D. Bumcrot
Hypothesis
B
S. aureus CXCR4 gRNAs
NNGRRT PAM 22-24mers
60
Outline
• Design/test S. aureus and S. pyogenes
gRNAs in 293T, K562, CD34+ cells
%Indels
60
S. aureus CCR5 gRNAs
NNGRRT PAM 22-24mers
40
40
20
20
0
0
Cas9
• Compare DNA, RNA, and RNP
delivery in human CD34+ cells
Genome editing at CXCR4 locus in
mobilized peripheral blood CD34+ cells
A
B
30
%Indels
%Indels
• Evaluate editing at target loci (T7E1
assay on locus PCR products), HSC
phenotype, function, viability
Multiplex genome editing
20
10
20
10
0
2
1
0
pmax
Sa
Sa
Spy
Spy
GFP CXCR4 CXCR4 CXCR4 CXCR4
+
+
FSC
CD90
7-AAD
CFCs
D
CD34
C
SSC
• Cas9/gRNA RNP delivery
20% editing in K562 cells, HSCs require
further development
Fold expansion
of CD34+ cells
• Cas9/gRNA DNA delivery
>20% genome multiplex genome editing
while maintaining HSC viability and
hematopoietic potential
3
Sa
Sa
Spy
Spy
CXCR4 CXCR4 CXCR4 CXCR4 Optimized
+
+
conditions
Annexin-V
CCR5 locus CXCR4 locus
Fold expansion
of CD34+ cells
Summary
0
3
2
1
0
unNucleofected control
Spy Cas9 +
CXCR4+CCR5 gRNAs
GEM
M
E
300
200
GM
M
100
G
0
no
electroporation
Sa CXCR4
Sp CXCR4
Sp CCR5
Sp CXCR4 +
CCR5
GEMM
E
AAVS1 locus
40
200
GM
M
G
100
20
0
K562
cells
B 75K562 cells
CD34+ CFCs
cells
0
Propidium Iodide
HBB locus
HBB_Sp8
50
HBB_Sp15
25
0
RNA
C25
(A) Percentage of indels
detected at CXCR4 locus 72
hours after electroporation of S.
aureus or S. pyogenes Cas9
and
paired
gRNAs
(CXCR4_Sp35
or
CXCR4_Sa18) in human CD34+
cells +/- optimized cell culture
conditions.
Bottom:
Fold
expansion of live CD34+ cells 72
hours after electroporation. (B)
Multiplex genome editing in
CD34+ cells after co-delivery of
S.
pyogenes
Cas9,
CXCR4_Sp35,
and
CCR5_Sp43 gRNA plasmids.
Bottom: Fold expansion of live
CD34+ cells 72 hours after
electroporation.
(C)
Representative flow cytometry
analysis of CD34+ cells cultured
in optimized conditions at 72
hours after Cas9/gRNA plasmid
delivery. (D) Ex vivo colony
forming potential of DNA
electroporated
CD34+
cells
cultured in optimal conditions.
untreated
control
AAVS1 gRNA
+ Cas9
Cord blood CD34+ cells
CXCR4_Sp35
AAVS1_Sp1
HBB_Sp8
20
15
10
5
0
RNP
FIGURE 2. Genome editing in human mobilized peripheral blood CD34+ cells after electroporation of
CAS9 and gRNA plasmid DNA.
30
• Cas9 mRNA/gRNA delivery
>25% editing while maintaining HSC
viability and hematopoietic potential
60
CFCs
0
0
300
2
D
µg gRNA
HBB
10
CXCR4
AAVS1
untreated control
Annexin V
20
20
A
Mobilized peripheral blood CD34+ cells
7-AAD
E
400
CFCs
40
FIGURE 3. Human CD34+ cells electroporated with S. pyogenes Cas9 mRNA and gRNA
maintain viability and multipotency and have sustained genome editing.
%Indels
40
(A) S. pyogenes Cas9 with
gRNAs targeting loci with
different
PAMs.
%
insertion/deletion
(nonhomologous end joining) on
the Y axis represents on-target
cleavage rates as measured
by T7E1 assay. (B) S. aureus
Cas9 with gRNAs targeting
loci with different PAMs. %
insertion/deletion (indels, nonhomologous end joining) on
the Y axis represents on-target
cleavage rates as measured
by T7E1 assay. Transfection
into HEK293T cells using
Lipofectamine 3000 in 24-well
format with 750 ng/well Cas9)
plasmid (CMV promoter) and
250 ng/well of gRNA (U6
promoter) constructs.
CD34
60
S. pyogenes CCR5 gRNAs
NGG PAM 17-20mers
%Indels
S. pyogenes CXCR4 gRNAs
NGG PAM 17-20mers
60
%Indels
• Optimization of culture and delivery
conditions to maintain CD34+ cell
survival and ex vivo hematopoietic
potential will facilitate development of
clinically beneficial levels of targeted
gene modification in HSPCs
A
%Indels
• Nonviral methods of Cas9/gRNA
delivery reduce CD34+ HSPC viability,
survival, multipotency
•
Optimize
conditions
and
components to maintain HSCs
Editas Medicine, Cambridge, MA 02142
FIGURE 1. Screening of S. pyogenes and S. aureus CXCR4 and CCR5 gRNAs.
GEMM
E
300
GM
200
M
100
G
0
untreated
control
2µg
HBB gRNA
10µg
HBB gRNA
CFU-GEMM
CFU-GM
CFU-E
CFU-M
(A) Percentage of indels
detected at AAVS1 locus 72
hours after electroporation of
S. pyogenes Cas9 and AAVS1
gRNA in K562 cells, mobilized
peripheral blood CD34+ cells,
and the progeny of edited
CD34+ cells (CFCs). Middle
panel: Representative flow
cytometry analysis of viable
(propidium iodide negative)
CD34+ cells. Right panel:
Hematopoietic colony forming
potential of unedited and
edited hematopoietic progeny.
(B) Genome editing at the
HBB locus in K562 cells after
S. pyogenes Cas9 mRNA or
Cas9 ribonucleoprotein codelivered with single HBB
gRNAs
(HB_Sp8
or
HBB_Sp15). (C) Percentage
of indels detected at the
indicated target genetic loci
after delivery of Cas9 mRNA
and gRNA to cord blood
CD34+
cells.
Right:
Representative gel showing
cleavage at the indicated loci
(T7E1 analysis). (D) Cell
viability in CB CD34+ cells 48
hours after delivery of Cas9
mRNA and indicated gRNAs
as determined by co-staining
with 7-AAD and Annexin V
and flow cyotometry analysis.
(E)
CFC
analysis
and
representative
colonies
generated from edited CB
CD34+ cells.
CFU-G