Ø Researchers used CRISPR/Cas9 nuclease and nickase to target and repair the c.456+4A>T mutation in the FANCC gene, which causes Fanconi anemia (FA), in human fibroblasts derived from an FA patient. Ø Both nuclease and nickase mediated repair of the mutation, restoring normal FANCC gene function, though nickase was more efficient due to preferentially inducing the homology-directed repair pathway. Ø Genome-wide analyses found no off-target effects, confirming the CRISPR reagents specifically targeted the intended FANCC locus. This provides proof-of-principle that CR

1. CRISPR/Cas9 as a Promising Gene Editing Tool for Fanconi Anemia Treatment
Vineeta Sharma10, Mark J. Osborn1-­3, Richard Gabriel4,5, Beau R. Webber1, Anthony P. DeFeo1, Amber N. McElroy1, Jordan Jarjour6, Colby G. Starker2,3, John E. Wagner1,3,
J. Keith Joung7,8
, Daniel F. Voytas2,9, Christof von Kalle4,5, Manfred Schmidt4,5, Bruce R. Blazar1,3, Mark J. Ahn10, Timothy J. Miller10, Jakub Tolar 1,3
1Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN 55455., 2Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455.
3Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455., 4Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg 69120, Germany. 5German Cancer Research Center (DKFZ), Heidelberg 69120, Germany.,
6Pregenen, Inc., Seattle, WA 98103., 7Molecular Pathology Unit, Center for Computational & Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02114.,
8Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115., 9Department of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN 55455.,10Abeona Therapeutics, Inc., Cleveland, OH 44103
Introduction: Fanconi anemia (FA) is a rare inherited disease
manifested by bone marrow failure and increased risk of malignancy.
The c.456 + 4A>T (IVS4 + 4A>T) point mutation in FA
complementation group C (FA-­C) gene results in a cryptic splice site
and causes aberrant splicing and in-­frame deletion of FANCC exon 4.
Gene editing is highly desirable alternative to allogeneic
hematopoietic cell transplantation (HCT) for FA. In the present study,
we have generated a CRISPR/Cas9 system for FANCC locus and
demonstrated its usefulness in repairing the FANCC c.456+4A>T
mutation.
Methods and Results: To test the ability of our custom-­designed
CRISPR/Cas9 reagents to mediate FANCC gene HDR, a transformed
skin fibroblast culture was derived from an FA-­C patient homozygous
for the c.456+4A>T mutation [1]. The cells were treated with a donor
plasmid and either the CRISPR/Cas9 nuclease or nickase. To
determine whether genome editing by CRISPR/Cas9 resulted in
restoration of exon 4 expression, HDR-­specific PCR was performed
using an allele specific RT-­PCR. Interestingly, CRISPR/Cas9 nuclease
and nickase clones each identified correction of c.456+4A>T
compared to untreated and WT controls. Furthermore, to evaluate
functional capability of our gene editing method, H2AX staining clearly
demonstrated inability of untreated FA-­C cells to phosphorylate γ-­
H2AX, however, the clones that were corrected by the nickase or the
nuclease showed clear evidence to phosphorylate γ-­H2AX. These
findings confirm correction of the c.456+4A>T mutation at DNA, RNA
and protein level.
An important safety concern of gene editing based correction strategy
is potential for off target (OT) effects. To assess this important safety
issue, a surveyor assay and an integration deficient lenti virus (IDLV)
reporter gene trapping assay was performed and no OT activity for the
nuclease or nickase was observed. Moreover, to identify the sites of
integration of the IDLV, the samples were tested using LAM PCR and
nonrestrictive (nr)LAM PCR, these results documented only on target
events. In total, the data suggests highly specific CRISPR/Cas9
reagents.
Conclusions: To summarize, this data show that CRISPR/Cas9
mediated direct c.456+4A>T mutation repair resulted in normalization
of the FANCC gene. This study also demonstrates that nickase was
more efficient and reliable compared to nuclease. Furthermore, the
gene editing model system established here provides support for a
favorable safety profile using these synthetic molecules for correction
of FA and other genetic disease in human cells.
ØWe have generated CRISPR/Cas9 nuclease and nickase reagents for targeting c.456+A>T mutation at FANCC locus.
ØOur data demonstrates both nuclease and nickase mediated c.456+A>T repair, however, the nickase was more efficient due to its
preference towards error free HDR pathway over NHEJ.
ØIn silico and genome wide LAM PCR methodologies confirmed highly specific on-­target HDR activity of our CAS9 reagents resulting in
phenotypic rescue of FANCC in an ex vivo disease model where fibroblasts were derived from a patient with FA.
Ø Aside from bone marrow transplantation that carries a risk of significant side effects, there is no treatment available that can halt or
reverse the symptoms of FA. Using the CRISPR-­Cas9 gene-­editing system to repair the FANCC gene in human fibroblasts from a FA
patient, our study has demonstrated significant and promising results.
Ø Our study provides proof of principle that CRISPR/Cas9 system has the potential to allow safe and precise gene modification for FA and
other blood disorders in human cells.
Cells
Human
NHEJ
InDel resulting in premature
Stop codon
HR
Homology directed recombination for
precise gene editing
Ex-­vivo
Cas9 RuvC
domain
Cas9 HNH
domain
Fig.1) The core components of CRISPR-­Cas9 are a nuclease Cas9
comprising two catalytic active domains RuvC and HNH, and a
guide RNA (gRNA). gRNA directs Cas9 to the target site by base-­
pairing, resulting in Cas9-­generated site-­specific DNA double-­
strand breaks (DSBs) that are subsequently repaired by
homologous directed repair (HDR) or by non-­homologous end-­
joining (NHEJ). Additionally, Cas9 can be reprogrammed into
nickase by inactivating either RuvC or HNH. Nickase makes single
stranded breaks and favors HDR over error prone NHEJ.
CRISPR can be used in seemingly for ex-­vivo or in-­vivo genome
editing.
Figure 1: Mechanism of CRISPR-­CAS9 mediated genome editing
Fig. 2-­A) CRISPR Design Tool identified a target site within 15 bp of c.456+4A>T
locus. B) CRISPR architecture and FANCC gene target recognition. C) Nuclease or
nickase were expressed form a plasmid containing the CMV promoter and BGH pA,
gRNA gene expression was mediated by U6 Poly IIIa promoter and a transcriptional
terminator (pT). D) The FANCC locus in cells that received CRISPR/Cas9 nuclease
or nickase with corresponding gRNA (target site shown as a green box), or a GFP-­
treated control group (labeled “C”), were amplified with primers (red arrows).
Nuclease-­ or nickase-­generated insertions/deletions result in heteroduplex
formation with unmodified amplicons that are cleaved by the Surveyor nuclease
resulting in 228 & 189 bp products. Surveyor assay indicated higher rates of
activity of nuclease compared to nickase. E) 293T cells F) FA-­C fibroblasts.
Figure 2: FANCC c.456+4A>T gene targeting
Figure 3: Assesment of DNA repair fates
Fig. 3) Quantification of NHEJ and HDR using TLR: A) At its basal state, the TLR
construct does not express a functional fluorescent protein, however, following
clevage of the target sequence in context to an exogenous GFP donor repair template,
GFP expression can be restored by HDR repair. Conversely, target site cleavage and
repair by the error-­prone NHEJ results in an in-­frame mCherry expression. B, C) Basal
rate of green or red fluresence were minute for either untransfected cells or cells
receiving donor only. D) Nuclease delievery resulted in both mCherry and GFP
fluorescence, indicating both NHEJ and HDR events, however, nickase version of
Cas9 promotes HDR and minimizes NHEJ.
Figure 4: Homology-­directed repair and phenotypic restoration
Fig. 4-­ i) To test the ability of our custom designed Cas9 reagents to mediate FANCC gene HDR, transformed skin fibroblast
culture from an FA-­C patient homozygous for the c.456+6A>T was used. i-­A) The FANCC locus, red arrow indicates
c.456+4A>T locus, blue arrow indiactes primers used for HDR screening. B) Gene correction Donor. C) Gel image of PCR
screening approach for HDR using donor and locus specific primers. D) Number of gene-­corrected clones obtained. E)
Sanger sequencing data of untreated cells and gene corrected clones confirms correction of c.456+4A>T mutation. B) Cas9
mediated HDR restores FANCC expression at mRNA and protein level in ex-­vivo culture system where fibroblasts were taken
from an FA-­C patient homozygous for c.456 A>T . ii, A-­E) CRISPR-­Cas9 mediated HDR of c.456 A>T restores FANCC
expression at DNA, mRNA and protein levels in patient fibroblast.
Figure 5: CRISPR off-­target and on-­target analysis
Fig. 5-­ i-­A) CRISPR Design Tool revelead five intragenic OT sites. i-­B) Surveyor analysis indicated no off-­target activity for
any of the five intragenic OT sites. ii-­ A) Tandam delivery of CRISPR/Cas9 nuclease or nickase with GFP IDLV resulted in GFP
expression. ii-­B) GFP expressed cells were sorted and expanded. ii-­C, D) PCR analysis using a 3′ LTR forward primer and a
FANCC locus reverse primer yielded a PCR product for the Cas9 nuclease and nickase treated cells but not the IDLV-­only
control cells. Sequencing of these products showed an LTR:FANCC genomic junction immediately upstream of the CRISPR
PAM (data not shown) suggesting high specificity of CAS9 reagents. Iii-­A, B) Genome-­wide screen for off-­target loci reported
CLIS frequency as 7-­31 at intended target loci, while no CLIS activity was reported at loci containing partial target site
homology.
1: Osborn, M.J., Gabriel, R., Webber, B.R., DeFeo, A.P., McElroy, A.N., Jarjour, J., Starker, C.G., Wagner, J.E., Joung, J.K., Voytas, D.F., von, Kalle. C., Schmidt, M., Blazar, B.R.,
Tolar, J. Fanconi anemia gene editing by the CRISPR/Cas9 system. Hum Gene Ther. 2015, 26(2):114-­26.
Technology is licensed by Abeona Therapeutics Inc.Funded by the National Center for Advancing Translational Sciences of the National Institute of Health Award Number UL1TR000114 (MJO).
i ii iii
iii
BACKGROUND
ABSTRACT RESULTS
CONCLUSIONS
REFRENCES
MATERIAL & METHODS
CRISPR & donor
construction
Gene Transfer
Surveyor
Nuclease
Selection &
transgene
excision
Traffic light
reporter cell
line generation
Cas9 nuclease & nickase
screening
Molecular &
protein
screening
Off-­target
analysis
Genome-­wide
screening