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Therapeutic Correction of an LCA-Causing Splice Defect
in the CEP290 Gene by CRISPR/Cas-Mediated Genome Editing
Morgan L. Maeder, Rina Mepani, Sebastian W. Gloskowski, Maxwell N. Skor, McKensie A. Collins, Gregory M. Gotta, Eugenio Marco, Luis A. Barrera, Hari Jayaram, David Bumcrot
ABSTRACT
RESULTS
Gene editing in LCA10 patient fibroblasts using
S. aureus Cas9 and two guide RNAs.
Molecular basis of disease
Wild-type CEP290
DNA
Intron 26
Exon 26
Exon 27
Transcription and splicing
mRNA
Exon 26
Correct CEP290 protein
Exon 27
Quantification of targeted genomic deletion in primary fibroblasts
transfected with Cas9 and gRNAs by droplet digital PCR.
25
20
DNA
TAGT
Exon 26
15
***
128bp
Exon 27
323+11
DNA
*
AATTGTGAGT
Exon 26
Exon 27
NHEJ
Edited DNA
Exon 26
Exon 27
Transcription and splicing
Exon 26
Exon 27
490+502
490+504
492+502 492+504
Sequence and
data analysis
PCR (both
directions)
GFP
0.0004
Target
Site
On-target editing
rate (% indels)
64
96.5
No off-targets identified
323
94.8
No off-targets identified
490
78.38
No off-targets identified
492
93.76
No off-targets identified
496
94.38
No off-targets identified
504
72.09
No off-targets identified
WT
Mut
0.0005
****
****
****
****
****
11
****
93.5
0.0003
0.0002
0.0001
0.0000
****
****
323+11
****
****
****
****
****
323+64 490+496 490+502 490+504 492+502 492+504
GFP
Figure 3: Increased CEP290 expression
Increased expression of wildtype CEP290 protein as
measured by Western blot in primary patient fibroblasts
transfected with Cas9 and gRNAs.
4
3
2
1
0
323+11
mRNA
490+496
Increased expression of wildtype transcript and decreased
expression of mutant transcript in primary patient fibroblasts
transfected with Cas9 and gRNAs as measured by qRT-PCR.
Cys998X
Gene Editing Strategy
323+64
Figure 2: Targeted deletion corrects splicing
Exon 27
Correct CEP290 protein
Isolate gDNA,
shear,
ligate barcoded
adapters
***
0
Fold change compared to GFP
Exon 27
***
5
mRNA
Exon 26
****
****
10
Transcription and splicing
Exon 26
34bp endprotected dsODN
****
IVS26 mutant CEP290 (c.2991 + 1655 A>G)
*
AATTGTGAGT
GUIDE-Seq in U2OS cells and primary patient fibroblasts, followed
by targeted amplicon sequencing reveals few off-target sites.
****
Relative mRNA
METHODS
Figure 1: Targeted genomic deletion
%Deletion
Leber congenital amaurosis (LCA) comprises a
genetically heterogeneous group of early-onset
retinal disorders characterized by severe loss of
vision in the first years of life. Approximately 20%
of LCA patients harbor mutations in the CEP290
gene, which exceeds the packaging limit of AAV
and is therefore not amenable to traditional gene
therapy. Here, we report a gene editing approach
in which the CRISPR/Cas9 system is used to
modify the endogenous CEP290 locus and restore
normal function of the gene.
Figure 4: Specificity profiling of candidate gRNAs
reveals few off-target sites
323+64 490+496 490+502 490+504 492+502 492+504
GFP
502
93.34
Off-Target
Site
Off-Target
Location
Off-target editing
rate (% indels)
Chr17:55416466
Intron, MMD
0.03
Chr2:10678496
Intron, NOL10
ND
Chr3:71041347
Exon, FOXP1
0.27
Chr4:17268500
intergenic
8.81
Chr10:46526823
intergenic
0.12
Chr2:119441891
Intron, SCTR
ND
Chr1:42755713
Intron, LEPRE1
ND
Chr4:97307689
intergenic
0.05
Chr1:247853709
intergenic
ND
Chr2:2526357
intergenic
0.12
CONCLUSIONS
This work supports the development of a gene-editing
approach for therapeutic treatment of CEP290-associated
disease caused by the IVS26 c.2991+1655 A>G mutation.
The use of the S. aureus CRISPR/Cas9 system enables
efficient packaging of the Cas9 gene, as well as two gRNA
genes, into a single AAV vector and provides a method for
delivery into patient photoreceptors.
Correct CEP290 protein
The patient fibroblasts were generously provided by Budd Tucker at the University of Iowa.
The authors declare competing financial interests. All authors are employees of Editas Medicine.