Download Read More - Editas Medicine

Screening S. aureus CRISPR-Cas9 Paired Guide RNAs for Efficient Targeted
Deletion in Duchenne Muscular Dystrophy
Josh Tycko1, Nick Huston1, Jacqueline Robinson-Hamm2, Chris Wilson1, Charles A. Gersbach2, Patrick D. Hsu1, David Bumcrot1
1. Editas Medicine, 300 Third St., Cambridge, MA 02142 2. Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708
Absolute quantification of deletions with digital droplet PCR
1000
80%
60%
40%
50
40
100
Guide RNA pairs
with a Z score > 1.5
were selected as
hits for validation
10
20%
1
0%
Female ΔExon 51
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
Guide Hit Validation
n=4
30
20
10
0
Guide Pair Rank
Z score
Male ΔExon 51 Male WT Exon 51
Deletion Efficiency (%)
Number of Guide Pairs
There is ~90 kb of intronic space
wherein targeted paired guide RNAs
(light blue) could mediate a deletion
of Exon 51.
120%
Observed Exon 51 Deletion
•  Thus, CRISPR/Cas9 targeted deletions that
restore the reading frame could convert
DMD genotypes into BMD-like genotypes
and potentially ameliorate the phenotype
of this disease. The smaller Cas9 ortholog
from S. aureus can be packaged with paired
guide RNAs in an AAV vector for in vivo
gene editing.
A multiplex Taqman ddPCR assay to
quantify Exon 51 deletion.
Experimental Overview
100%
Deletion size is one
factor affecting
paired guide RNA
deletion efficiency.
80%
60%
y = 0.915x + 0.0744
R² = 0.99122
40%
20%
0%
0%
20%
40%
60%
80%
100%
Expected Exon 51 Deletion
•  Deleting Exon 51 would restore the
dystrophin reading frame for the greatest
number of patients of any single exon
deletion.
Deletion efficiency of 857 SaCas9 paired guide RNAs
Activity of lead candidate guide RNA pairs confirmed
Conclusions
Human-only guide RNA pairs
Deletions <14kb
Human-NHP cross reactive guide RNA pairs
Deletions >14kb
•  Screening identified several candidate
guide RNA pairs, including pairs with up to
25% deletion efficiency in DMD myoblasts.
0
-5
-10
Guide Pair Rank
Upstream Guide RNA
← Distance from Exon 51
Deletion Efficiency (%)
15
5
30
20
10
0
12 lead guide RNA pairs were validated
by plasmid transfection in HEK293T cells.
30
20
10
0
A subset of leads were electroporated
into immortalized DMD myoblasts.
Distance from Exon 51 →
20
10
40
Deletion Efficiency (%)
Guide RNAs were cloned into
plasmids and transfected into
HEK293T cells along with an SaCas9
plasmid. Exon 51 deletion efficiency
was measured after 3 days by
ddPCR.
Deletion Efficiency (%)
40
•  From the set of 10,553 21-nt S.aureus Cas9
(SaCas9) guide RNAs targeting human DMD
introns 50 and 51, we selected pairs that
passed design filters intended to improve
guide RNA expression, improve SaCas9
cleavage efficiency, and minimize off-target
editing concerns.
•  Deletion size plays a role in deletion
efficiency, but is not highly predictive.
All Guide Pairs
Positive Control Guide Pair
Non-targeting Guide
100%
•  In contrast, Becker muscular dystrophy
(BMD) patients generally have mutations in
dystrophin that do not disrupt the reading
frame, leading to a milder disease
phenotype.
•  Lead guide RNAs mediate the precise
desired deletion in immortalized DMD
myoblasts.
Screen analysis and validation of 78 hits
120%
•  Duchenne muscular dystrophy (DMD) is a
X-linked recessive neuromuscular disorder
that results in progressive muscle
degeneration and premature death. Most
patients have frameshifting mutations in
the dystrophin gene that result in a
nonfunctional protein.
Exon 51 Deletion
Background
68
69
70
71
72
73
74
75
76
77
78
79
80
81
Downstream Guide RNA
82
83
84
85
86
87
88
89
90
91
92
93
23.7
Max
9.5 Average
-2.3
Min
Pos Ctrl Avg
16.9
Neg Ctrl Avg
SD
2.1
SD
2.3
2.4
Intron 50
Intron 51
Sequencing one of the immortalized DMD myoblast samples showed that a
pair of guide RNAs can mediate the precise expected deletion.