CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease

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Introduction
Expansion of a CAG/glutamine repeat in various genes causes at
least 9 different neurodegenerative diseases, including Huntington’s
disease (HD). In HD, the expanded CAG repeat encodes a polyglutamine (polyQ) tract in the N-terminal region of huntingtin (HTT)
and leads to a wide range of cellular dysfunctions (1). The gain of
toxic function of mutant huntingtin (mHTT) has led to considerable
efforts to use siRNA, antisense oligonucleotides, or CRISPR/Cas9 to
selectively suppress the expression of mHTT (2–4). Indeed, siRNA
and antisense oligonucleotides have shown promising therapeutic
effects in HD mice that express transgenic mHTT (2, 5). However,
this relies on SNPs that are specific to the mutant allele. Also, whether this strategy can be successfully used in HD mice that express
mHTT at the endogenous level remains unknown, since normal and
expanded alleles of the endogenous Htt gene are not readily distinguished by siRNA and antisense oligonucleotides (6).
Our recent studies using conditional Htt-KO mice revealed that
depletion of normal HTT in adult mouse brains does not affect animal survival, growth, or neuronal viability (7). In addition, knockin
(KI) mice that express N-terminal mHTT have shown that the N-terminal region of HTT is not essential for early embryonic development (8). These findings suggest that removal of N-terminal HTT
containing the polyQ domain, regardless of its allele, could be a
potential therapeutic strategy to treat HD. Here, we report that permanent suppression of the endogenous expression of mHTT via
CRISPR/Cas9 in the striatum of HD140Q-KI mice, which express a
human HD HTT, can effectively deplete HTT aggregates and early
neuropathology, even after the formation of abundant HTT aggregates. Reducing HTT expression in striatal neuronal cells does not
affect the viability of the adult HD140Q-KI mice, but alleviates their
motor deficits and neurological symptoms. Our findings suggest that
depletion of HTT via CRISPR/Cas9 in a non–allele-specific manner
can efficiently and permanently eliminate polyQ expansion–mediated neuronal toxicity in the adult brain. This also opens up a new
avenue for treating other neurodegenerative diseases caused by the
gain-of-function mechanism.
Results and Discussion
To delete the polyQ domain of mHTT using CRISPR/Cas9, we
designed 4 guide RNAs (gRNAs) to target the DNA regions (T1,
T2, T3, and T4) flanking the CAG repeat in exon 1 of human HTT
(Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/
JCI92087DS1). We transfected HEK293 cells stably expressing
exon 1 of human HTT containing 120 CAG repeats with each of
the 4 gRNAs and Cas9. Western blotting showed a reduction of
mHTT in the transfected cells (Supplemental Figure 1, B and C).
We also tested the activities of combining 2 HTT-gRNAs in the stable HEK293 cells. A combination of T1 and T3 HTT-gRNAs led to
the greatest reduction in mHTT (Supplemental Figure 1, D and E)
and was used for our subsequent studies.
We next tested the effect of CRISPR/Cas9-mediated HTT
depletion in HD140Q-KI mice. In this KI mouse model, exon 1
of human HTT with 140 CAG repeats replaces exon 1 of endogHuntington’s disease is a neurodegenerative disorder caused by a polyglutamine repeat in the Huntingtin gene (HTT).
Although suppressing the expression of mutant HTT (mHTT) has been explored as a therapeutic strategy to treat
Huntington’s disease, considerable efforts have gone into developing allele-specific suppression of mHTT expression, given
that loss of Htt in mice can lead to embryonic lethality. It remains unknown whether depletion of HTT in the adult brain,
regardless of its allele, could be a safe therapy. Here, we report that permanent suppression of endogenous mHTT expression
in the striatum of mHTT-expressing mice (HD140Q-knockin mice) using CRISPR/Cas9-mediated inactivation effectively
depleted HTT aggregates and attenuated early neuropathology. The reduction of mHTT expression in striatal neuronal cells in
adult HD140Q-knockin mice did not affect viability, but alleviated motor deficits. Our studies suggest that non–allele-specific
CRISPR/Cas9-mediated gene editing could be used to efficiently and permanently eliminate polyglutamine expansion–
mediated neuronal toxicity in the adult brain.
CRISPR/Cas9-mediated gene editing ameliorates
neurotoxicity in mouse model of Huntington’s disease
Su Yang,1
Renbao Chang,1,2,3 Huiming Yang,1
Ting Zhao,1
Yan Hong,1
Ha Eun Kong,1
Xiaobo Sun,4
Zhaohui Qin,5
Peng Jin,1
Shihua Li,1
and Xiao-Jiang Li1,6
1
Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA. 2
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. 3
University of
Chinese Academy of Sciences, Beijing, China. 4
Department of Mathematics and Computer Sciences, and 5
Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University,
Atlanta, Georgia, USA. 6
Guangdong-Hongkong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, China.
Authorship note: S. Yang and R. Chang contributed equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: December 2, 2016; Accepted: May 4, 2017.
Reference information: J Clin Invest. 2017;127(7):2719–2724.
https://doi.org/10.1172/JCI92087.
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Western blotting verified that RFP and Cas9 were predominantly
expressed in the injected striatum (Supplemental Figure 2B).
We injected AAV-HTT-gRNA and AAV-CMV-Cas9 into one
side of the striatum in homozygous HD140Q-KI mice at the age
of 3 or 9 months. The contralateral striatum was injected with
AAV-HTT-gRNA or AAV-CMV-Cas9 alone, which allowed us to
rigorously examine the efficiency of HTT-gRNA/Cas9–mediated mHTT knockdown. HD140Q-KI mice are known to develop
age-dependent motor deficits and nuclear accumulation of mHTT
(9, 14). We found that most of the striatum and the needle pathway in the cortex and hippocampus were transduced by AAVs 3
weeks after injection (Figure 1B). Western blotting showed that
enous mouse Htt (9), resulting in the expression of full-length
mHTT with 140Q under the control of the endogenous mouse Htt
promoter. In HD140Q-KI mice, accumulated mHTT in striatal
neuronal nuclei is detectable between 4 and 6 months and forms
obvious aggregates at 9 to 10 months (8, 10–12). We focused on
the striatum to investigate the effect of removing mHTT. Two
gRNAs (T1 and T3) are expressed under the U6 promoter in an
adeno-associated virus (AAV) vector that also expresses red fluorescent protein (RFP) (AAV-HTT-gRNA), and Cas9 is expressed in
another AAV vector under the CMV promoter (AAV-CMV-Cas9)
(13). The 2 viruses were mixed at a ratio of 1:4 for stereotaxic injection into mouse striatum (Supplemental Figure 2A). After 3 weeks,
Figure 1. CRISPR/Cas9 depletes the ubiquitous expression of mHTT in homozygous HD140Q-KI mouse striatum. (A) Schematics of the designed HTT-gRNA
(T1 and T3). (B) Immunofluorescence showing the transduction of AAV-HTT-gRNA in the striatum and part of the cortex. Ctx, cortex; Str, striatum; CC, corpus
callosum; LV, lateral ventricle. Scale bar: 100 μm. (C) Different brain regions from 9-month-old homozygous HD140Q-KI mice injected with AAV-CMV-Cas9 and
AAV-HTT-gRNA (T1 and T3) or control-gRNA were analyzed by Western blotting with 1C2 for mHTT and antibodies against Cas9, GFAP, NeuN, p62, caspase 3, and
cleaved caspase 3. Vinculin was used as a loading control. Hip, hippocampus. (D) Low- and high-magnification images show the reduction of nuclear HTT and HTT
aggregates in the AAV-HTT-gRNA/AAV-CMV-Cas9–injected area in 9-month-old homozygous HD140Q-KI mice compared with the contralateral striatum injected
with AAV-HTT-gRNA only. Arrow indicates a remaining cell with nuclear HTT inclusion. Scale bar: 10 μm. The red dashed outline indicates the injected region where
mHTT aggregates are markedly reduced. (E) Double immunostaining confirmed the depletion of mHTT in the area expressing HTT-gRNA in the injected striatum
of 9-month-old homozygous HD140Q-KI mice. The striatum of a HD140Q-KI mouse injected with AAV-CMV-Cas9 only was used as a control. Scale bar: 20 μm.
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astrocytes. We also checked several other proteins such as NeuN
(a neuronal marker), p62 (an autophagy marker) and caspase 3 (an
apoptosis marker), which are frequently studied in neurodegenerative diseases, and found that these proteins remained unchanged
(Figure 1C and Supplemental Figure 2C). The results were corroborated by immunohistochemical studies using GFAP and NeuN
antibodies (Supplemental Figure 3, A and B).
Most HD patients are heterozygous for the HD gene mutation. Also, neurons are preferentially affected in HD. Thus, we
tested the therapeutic potential of CRISPR/Cas9 in heterozygous
HD140Q-KI mice, using AAV-HTT-gRNAs (T1 and T3) with AAVCas9 that was expressed under the neuronal methyl-CpG–binding
protein (Mecp2) promoter (AAV-MECP2-Cas9) (Figure 2A). As a
control, AAV-control-gRNA with AAV-MECP2-Cas9 were used.
These viruses were mixed at a ratio of 1:4 (gRNA/Cas9) and injectHTT-gRNA, but not control-gRNA, caused a significant reduction of mHTT in the striatum of 9-month-old HD140Q-KI mice
(Figure 1C and Supplemental Figure 2C). Compared with the
contralateral striatum injected with HTT-gRNA alone, immunostaining revealed a dramatic decrease in the nuclear accumulation
and aggregation of mHTT in the HTT-gRNA/Cas9–injected striatum (Figure 1D). Double immunofluorescence staining further
verified that the decrease in mHTT staining is dependent on the
expression of HTT-gRNA (Figure 1E). In HD KI mouse brain, a
well-known early neuropathology is reactive astrocytes (15, 16).
In brain regions transduced by HTT-gRNA/Cas9, attenuation of
the increased glial fibrillary acidic protein (GFAP) was associated
with knockdown of mHTT compared with brain regions injected with control-gRNA/Cas9 (Figure 1C and Supplemental Figure 2C), indicating that a reduction of mHTT alleviated reactive
Figure 2. Behavioral analysis of heterozygous HD140Q-KI mice with depletion of neuronal HTT in the striatum by AAV-HTT-gRNA/AAV-MECP2-Cas9 injection.
(A) Schematics showing the viral vectors used. HA, human influenza hemagglutinin; ITR, inverted terminal repeat; KASH, Klarsicht, ANC-1, Syne Homology;
WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (B) Double immunostaining with anti–DARRP-32 indicated that medium spiny neurons
expressed AAV-HTT-gRNA. Scale bar: 10 μm. (C) Motor functions of heterozygous HD140Q-KI mice injected with AAV-HTT-gRNA/AAV-MECP2-Cas9 (KI HTT-gRNA)
or AAV-control-gRNA/AAV-MECP2-Cas9 (KI control-gRNA) and WT mice injected with AAV-control-gRNA/AAV-MECP2-Cas9 (WT) were evaluated using rotarod,
balance beam, and grip strength tests at different time points after injection (n = 12 for each group; *P < 0.05, **P < 0.012, and ***P < 0.001, by 2-way ANOVA
with Bonferroni’s test, comparing the KI HTT-gRNA group with the KI control-gRNA group).Data represent the mean ± SEM.
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KI mice. We were able to obtain 24 nine-month-old heterozygous KI
mice for examination, at which age the mice show abundant nuclear
accumulation of mHTT in striatal neurons and also develop obvious motor dysfunction. These KI mice were injected with either
AAV-HTT-gRNA/AAV-MECP2-Cas9 or AAV-control-gRNA/AAVMECP2-Cas9, and their behaviors were monitored for 3 months.
In HD140Q-KI mice, motor dysfunction has been well documented using rotarod, balance beam, and grip strength tests (9, 17). We
found that HTT-gRNA/Cas9 could significantly improve performance in these tests and alleviate the motor deficits of HD140Q-KI
ed into both sides of the striatum of 9-month-old heterozygous
HD140Q-KI mice to maximize the therapeutic effects. Immunostaining of the injected striatum revealed the presence of RFP in
dopamine- and cAMP-regulated phosphoprotein as well as dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32)
(Figure 2B) and NeuN-positive (Supplemental Figure 4) neurons,
indicating that medium spiny neurons in the striatum had been
transduced by the injected AAVs.
We examined whether CRISPR/Cas9-mediated neuronal
mHTT depletion had any impact on the motor function of HD140QFigure 3. Removal of mHTT in neuronal cells alleviates neuropathology in 13-month-old heterozygous HD140Q-KI mouse striatum. (A) Western blotting
shows the reduction of mHTT in brain tissues from 3 heterozygous HD140Q-KI (KI-1, KI-2, and KI-3) and WT mice. 2166 Antibody was used to show both
mHTT and WT HTT. 1C2 Antibody was used to show only mHTT. Replicate samples run on separated blots are presented. (B) Double immunostaining with
1C2 antibody confirmed the depletion of mHTT by AAV-HTT-gRNA. A heterozygous HD140Q-KI mouse injected with AAV-control-gRNA served as a control.
Scale bar: 20 μm. (C) Quantitative assessments of the relative ratio of mHTT to total HTT in A (left; n = 8; ***P < 0.001, by 1-way ANOVA with Tukey’s
test) and relative levels of mHTT staining in B (right; n = 8; ***P < 0.001, by Student’s t test). (D) Double immunostaining of striatum (from 9-month-old
injected mice examined at 13 months of age) shows decreased GFAP levels by HTT-gRNA compared with control-gRNA. There was no difference in NeuN
staining. Scale bars: 20 μm. (E) Quantitative assessment of the relative levels of GFAP and NeuN staining (n = 8). The staining intensity for each mouse
was the average from three ×10 images. ***P < 0.001, by 1-way ANOVA with Tukey’s test. Data represent the mean ± SEM.
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obvious motor deficit phenotypes (8, 10–12). Our findings revealed
that CRISPR/Cas9-mediated gene inactivation could reverse the
neuropathology and behavioral phenotypes, even when the mice
were 9 months old, suggesting that old neuronal cells still have the
ability to clear the accumulated mutant proteins and repair early
injury once the expression of mutant proteins is blocked. Thus,
reducing mHTT expression in the brains of elder HD patients
might still be effective in alleviating neurological symptoms.
Given that CRISPR/Cas9 can permanently eliminate the
expression of targeted genes, using CRISPR/Cas9 should more
efficiently deplete the expression of mHTT than has been possible
with previous therapeutic approaches, which require continuous
administration. Also, the severe neurological symptoms of many
neurodegenerative diseases are often associated with the preferential vulnerability of selective neuronal populations. The use of
specific promoters allows CRISPR/Cas9 to target specific types
of neurons. Thus, using CRISPR/Cas9 to inhibit mutant protein
expression in specific brain regions opens up a new avenue for
treating HD as well as other neurodegenerative diseases that are
caused by a toxic gain of function of mutant genes.
Methods
Study approval. All procedures were performed in accordance with
NIH guidelines and the US Public Health Service’s Guide for the Care
and Use of Laboratory Animals and were approved by the IACUC of
Emory University, which is accredited by the American Association for
Accreditation of Laboratory Care (AAALC).
Whole-genome sequencing. Whole-genome sequencing data have
been deposited in the NCBI’s Sequence Read Archive (SRA accession
number SRP105422).
Statistics. Statistical significance was determined by 2-tailed Student’s t test, 1-way ANOVA, or 2-way ANOVA using GraphPad Prism
5.0 (GraphPad Software). A P value of less than 0.05 was considered
statistically significant.
Author contributions
SY, RC, SL, and XJL designed the study. SY, RC, HY, TZ, and YH,
performed experiments and collected the data, HEK, XS, ZQ, and
PJ performed whole-genome sequencing–related work. SY, RC, SL,
and XJL analyzed the data. SY, RC, and XJL wrote the manuscript.
Acknowledgments
This work was supported by grants from the NIH (NS036232 and
NS101701, to XJL, and NS095279, to SHL) and the National Natural
Science Foundation of China (grant 91332206).
Address correspondence to: Xiao-Jiang Li or Shihua Li, 347 Whitehead Building, 615 Michael Street, Atlanta, Georgia 30322, USA.
Phone: 404.727.3290; Email: xli2@emory.edu (X.J. Li); Phone:
404.712.2304; Email: sli@emory.edu (S. Li).
mice compared with control-gRNA/Cas9–injected KI and WT mice
(Figure 2C). In addition, knocking down HTT expression also attenuated body weight reductions (Figure 2C). The efficiency of mHTT
reduction in the striatum varied among the individual mice tested
(Supplemental Figure 5A). We found that the percentage of mHTT
reduction in each mouse correlated with its rotarod and balance
beam performance (Supplemental Figure 5, B and C).
Many studies have shown rare off-targets when specific gRNAs
are used (18, 19). Whole-genome sequencing analysis using genomic DNA extracted from HTT-gRNA/Cas9–injected striatum verified that genomic mutations predominantly occurred around the
HTT-gRNA targeted sequences in the Htt gene, but not in potential
off-target loci (Supplemental Figure 6). This result was further corroborated by a T7E1 assay showing a lack of DNA mutations in selected potential off-target loci (Supplemental Figure 7A). In addition,
DNA sequencing confirmed the presence of frameshift mutations
around the targeted region of the HTT-gRNA (Supplemental Figure
7B). Western blotting analysis of individual injected mice showed an
obvious reduction of mHTT in the striatum and part of the cortex
containing the injection pathway compared with mHTT levels in the
hippocampus (Figure 3, A and C). Double immunostaining confirmed
a specific reduction of mHTT by HTT-gRNA, but not control-gRNA,
in the injected striatal area (Figure 3, B and C). Furthermore, AAVMCEP2-Cas9, which selectively depleted HTT in neuronal cells,
could also reduce reactive astrocytes but did not alter expression of
the neuronal marker NeuN (Figure 3, D and E). We also examined
striatal volume and brain weight and did not find significant differences between HTT-gRNA– and control-gRNA–injected HD140QKI mice (Supplemental Figure 8). These results suggest that neuronal
mHTT causes early neuropathology to increase reactive astrocytes in
HD140Q-KI mouse brains, which could be diminished by eliminating the expression of HTT. Taken together, removal of endogenous
HTT in neuronal cells via CRISPR/Cas9 can efficiently alleviate
mHTT-mediated neuropathology in HD140Q-KI mice.
Although shutting off the expression of transgenic mHTT can
alleviate neurological symptoms in HD mice (20, 21), whether
reducing the expression of endogenous HTT can be used to treat
HD without deleterious effects remains unknown. When we used
homozygous HD140Q-KI mice in which 2 alleles of the mHTT
gene could be disrupted by CRISPR/Cas9, we found that removal
of HTT did not affect the expression of NeuN or caspase 3. Instead,
mHTT depletion significantly reduced reactive astrocytes, an early pathological event in HD KI mouse brains (15, 16). The results
also support our recent findings that depletion of endogenous
mouse HTT in adult neurons is nondeleterious and that the function of HTT is cell type and age dependent (7). Using heterozygous
HD140Q-KI mice to further analyze their pathology and behaviors, we also verified that CRISPR/Cas9 could effectively alleviate
HD-related phenotypes. At the age of 9 months, HD140Q-KI mice
show abundant nuclear mHTT accumulation in the striatum and
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