DNA double-strand break-free CRISPR interference delays Huntington’s disease progression in mice

Plasmids

LentiCRISPRv2 (Addgene; 52961) was purchased from Addgene (Cambridge, MA, USA). For HTT gene suppression, LentiCRISPR v2 plasmid with U6 promoter driving sgRNA expression and EF-1 alpha promoter driving the expression of Puromycin-T2A-HA-NLS-dCas9-NLS (or Puromycin-T2A-Flag-NLS-Cas9-NLS) was used. The sgRNA-Cas9 and sgRNA-dCas9 constructs were generated by cloning the CAG repeat-targeting sgRNA sequences into a lentiCRISPRv2 containing puromycin resistance. The sgRNA-dCas9 construct was generated by mutating the Cas9 sequence in lentiCRISPRv2 to include D10A and H840A mutations, which result in catalytic inactivation of Cas9. The Cas9 only construct was generated by single CRISPR-Cas9 vector infections. Spacer sequence of sgRNA to target the CAG repeat region showed in Supplementary Table 1.

Lentivirus production

HEK293T cells (ATCC, CRL-11268) were cultured in 10-cm tissue culture dishes until they reached 70–80% confluence, after which plasmids that contained the gene of interest (10 μg), psPAX2, and pMD2G (5 μg and 2.5 μg, respectively), were transfected at a ratio of 4:2:1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 8 h of transfection, the culture medium was exchanged with 12 ml of growth medium. The viral supernatants were collected 48 h after the initial transfection and filtered through a Millex-HV 0.45 μm low-protein-binding membrane (Millipore, Darmstadt, Germany) before being concentrated 1000-fold by ultracentrifugation (Optima XPN-100 Ultracentrifuge, Beckman Coulter) at 4 °C at 25,000 rpm for 2 h. The pellets were resuspended in phosphate-buffered saline (PBS) or growth medium. The titer of lentivirus was measured with Lenti-X p24 Rapid Titer Kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. The estimated infectious titers pf Cas9-only, Cas9-sgRNA, and dCas9-sgRNA viruses were 2.34 × 1010, 2.24 × 1010, and 2.20 × 1010 TU/ml, respectively. In addition, lentiviral vector constructs encoding Cas9-only (1.16 × 109 TU/ml), Cas9-sgRNA (1.28 × 109 TU/ml), and Cas9-sgRNA (1.02 × 109 TU/ml) with CMV promoter were from VectorBuilder (Santa Clara, CA, USA).

Cell culture

HEK293T cells (ATCC, CRL-11268) were cultured in Dulbecco’s modified Eagle media (DMEM; Hyclone, Irvine, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco) in an incubator (37 °C, 5% CO2). The cells were seeded for transfection in six-well plates at 8 × 104 cells per well. The HEK293T cells were transfected with 2 μg/well (six-well plate) of DNA using Lipofectamine (Invitrogen) for 6 h.

The HD fibroblast lines (GM09197, GM21756, GM04022, and GM04855, Coriell, Camden, NJ), which was derived from humans with HD, and normal fibroblast lines (GM04775 and GM07492, Coriell), were used in this study. The cells were grown in DMEM (Hyclone) supplemented with 15% heat inactivated FBS, 1% penicillin/streptomycin, and 0.1% MEM nonessential amino acids (MEM NEAA; Gibco) at 37 °C with 5% CO2. The cells were seeded for transduction in six-well plates at 5 × 104 cells per well. Cells were transduced with Cas9-sgRNA, dCas9-sgRNA, or Cas9 only. During transduction, the cationic polymer polybrene (8 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added to the viral media (Multiplicity of infection, MOI = 10). The medium was exchanged to remove the virus the day after transduction. To select cells transduced with the lentivirus constructs, which contain a puromycin-resistance gene, we treated the mixture with 2 μg/ml puromycin (Gibco) until the non-transduced cells (control) were completely dead.

Reporter cloning

To confirm the activity of sgRNA (CAG PAM), oligonucleotides containing target sites with CAG PAM were synthesized. The sequence of this insert fragment, with the target site shown in bold and the CAG PAM are in italics (forward; AATTCAGCAGCAGCAGCAGCAGCAGCAG, reverse; GATCCTGCTGCTGCTGCTGCTGCTGCTG) and annealed in vitro using a thermocycler (95 °C for 5 min and then ramped down to 25 °C at 5 °C per min). The annealed oligonucleotides were ligated into the reporter vectors digested with EcoR1 and BamH1. And to confirm the activity of Cas9-sgRNA for a target site with 27 CAG repeats, 27 CAG repeat sequence was PCR amplified using the PrimeSTAR polymerase (TAKARA, Tokyo, Japan). Next, the amplicons, using an NEBuilderHiFi DNA assembly kit (New England BioLabs GmbH, Frankfurt, Germany), were assembled with the linearized reporter plasmid at 50 °C for 30 min. After incubation, the products were purified using a MEGA quick-spin total fragment DNA purification kit (iNtRON Biotechnology, Korea) and transformed into competent cells.

Flow cytometry

Three days after transfection, Adherent HEK293T cells co-transfected with reporter plasmid were trypsinized and resuspended in 2% FBS in PBS. Single-cell suspensions were analyzed using the FACSAria II cell sorter (BD Biosciences, San Jose, CA, USA). Untransfected cells and cells transfected with either mRFP vector alone or eGFP vector alone were used as controls.

Deep sequencing

Five days after transfection, Adherent HEK293T cells co-transfected reporter plasmid (27 CAG repeats) were trypsinized and genomic DNA was extracted from pelleted cells using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA). The target sequences within reporter plasmid were PCR amplified using the PrimeSTAR polymerase (TAKARA) for the Cas9-sgRNA mediated cleavage experiment. We used 500 μg of genomic DNA per sample as a template for the first PCR. The PCR products were purified with a MEGAquickspin Total Fragment DNA Purification kit (iNtRON Biotechnology). For the second PCR, 100 ng of purified PCR products from the first amplification were annealed with an Illumina adapter and barcode sequences. The resulting products were isolated, purified, mixed, and analyzed by paired-end read sequencing using Illumina MiniSeq.

Off-target analysis

Potential off-target sites for sgRNA sequences in the human genome were determined by the Cas9-OFFinder algorithm previously described40. To determine the relative expression of sequences containing potential off-target sites, human HD fibroblasts were transduced with a lentivirus encoding dCas9-sgRNA cassette. One week after transduction, total RNA was extracted using a RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed using PrimeScript™ RT Master Mix (TAKARA). The gene qRT-PCRs were performed using SYBR Green (Applied Biosystems, Carlsbad, CA, USA) with the primers listed in Supplementary Table 1. The results of on-target sites (HTT) and off-target binding sites including ABHD11, ATN1, ATXN3, DCP1B, FOXJ2, FZD1, KMT2D, PLEC, SATB1, ZNF384, ZNF395, and ZNF853 were normalized to GAPDH expression levels. Several genes, such as ATXN1, ATN1, ATXN7, CACNA1A, and TBP, were additionally identified for off-target effects. All experiments were performed in triplicate.

Ethics approval

All mice were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and were kept under controlled climate conditions with a 12-h light/dark cycle and were provided standard food and water ad libitum. All experimental procedures and animal care were approved by the Yonsei University Health System Institutional Animal Care and Use Committee (YUHS-IACUC approval No: 2015-0396, 2019-0100) and all procedures comply with the IACUC guidelines.

Mice and stereotaxic injection

In this study we used a total of thirty R6/2 mice, a transgenic mouse model of HD that expresses a version of exon 1 of the human HTT gene that includes 160 ± 5 CAG repeats35. For stereotaxic injection, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal (i.p.) injection and were randomly assigned to three groups for treatment with a lentiviral construct (Cas9 only, n = 8; Cas9-sgRNA, n = 14; dCas9-sgRNA, n = 8) at postnatal week 4 (P28). The mice received a lentiviral construct with 2 × 106–107 TU/μl or PBS (2 μl each) in both striata using stereotaxic coordinates (AP + 0.4 mm from bregma; ML ± 1.9 mm from bregma; DV −3.3 mm from dura). Body temperatures were maintained at 37 °C in a heating chamber until the mice fully awakened.

To provide insight into the effects of the lentiviral construct or Cas9 expression in R6/2 mice, we performed additional experiments as to the matched cohort of PBS group (PBS, n = 11). This study was also to confirm the effects of the lentiviral construct in R6/2 mice at 8 weeks of age: Cas9 only (n = 4), Cas9-sgRNA (n = 4), and dCas9-sgRNA (n = 4). In addition, to investigate that CRISPRi or CRISPR-Cas9 system has any effect on wild-type mice in the absence of HD, we established new cohorts of 25 wild-type mice (12 weeks old): untreated (n = 8), Cas9 only (n = 5), Cas9-sgRNA (n = 5), and dCas9-sgRNA (n = 7); twelve wild-type mice (8 weeks old): Cas9 only (n = 4), Cas9-sgRNA (n = 4), and dCas9-sgRNA (n = 4).

Rotarod performance

The rotarod (No. 47600; UGO Basile, Comerio, VA, Italy) test was used to assess motor coordination and balance at constant speed (12 rpm and 16 rpm) and accelerating speed (4–40 rpm). The latency period before mice fell from the rod was measured twice during each test, and if the interval between two recorded scores was more than 60 s, the latency period was measured once more. Individual tests were terminated at a maximum latency of 300 s.

Open field activity test

Locomotor activity tracing was performed in an open field arena (30 × 30 × 30 cm). Mice were placed individually into the arena and monitored with a video camera. The open field box was cleaned with 70% ethyl alcohol and air-dried prior to the commencement of each trial for every mouse. The resulting data were analyzed using the video tracking system Smart Vision 2.5.21 (Panlab, Barcelona, Spain). In the tracking system, the floor of the open field is divided into 16 spaces (4 × 4). The distance of movement was assessed for 5 min.

RNA extraction

Total RNA was extracted from the cells and striata of mouse brains using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ protocols. Extracted RNA was quantified RNA concentrations with an A260/A280 ratio for quality control analysis using a NanoDrop 2000 (Thermo Fisher Scientific).

Quantitative real-time PCR (qRT-PCR)

cDNA synthesis was performed using Prime Script™ Reverse Transcriptase (TAKARA). Quantitative PCR reactions were prepared with SYBR Green (Invitrogen) according to the manufacturer’s protocol. Reactions were run on a Light Cycler thermal cycler (Roche Diagnostics, Mannheim, Germany) with the oligonucleotide primers reported in Supplementary Table 1. The results are expressed as fold-increase in mRNA expression of the gene of interest normalized to Gapdh (or GAPDH) expression by the ΔΔCt method. All experiments were performed in triplicate.

mRNA sequencing

RNA sequencing was processed for transcriptome analysis by Macrogen, Inc. (Seoul, Korea) as previously described41,42. The extracted total RNA of mouse brain samples was transcribed into a library of templates that can suitable for cluster generation using the TruSeq RNA Sample Preparation Kit (Illumina). Next, the transcriptome analysis was performed by follow procedures: Total 1000 ng RNA were selective and purified primary mRNA (poly-adenylated RNA) using magnetic beads to conjugate oligo-dT. In order to convert this mRNA into single-stranded cDNA, reverse transcriptase and random hexamer primers were used, and DNA-dependent synthesis of the second strand was suppressed by adding Actinomycin D. After removing the RNA template, double-stranded cDNA was synthesized by adding deoxyribolidine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP). And then, a single A base was added on the 3′ end of the adapter with a single T base overhang, and the amount of the sequence-ready library was amplified by polymerase chain reaction using adapter-ligation cDNA. During this amplification process, the polymerase stalls when it encounters a U base, the second strand becomes a bad template, and the amplified material retain information through one strand. The final cDNA library was quantified by qPCR (Kapa Library Quant Kit; Kapa Biosystems, Wilmington, MA) and normalized to 2 nmol/L for sequencing.

Digenome sequencing

As presented in Kim et al., intact genomic DNA and Cas9-digested genomic DNA was sequenced to depth of 30x to 40x by Macrogen, Inc. (Seoul, Korea) using whole-genome sequencing (WGS)43. The WGS data were used to assign a DNA cleavage score to each nucleotide position in the entire genome as previously described39 and the DNA cleavage site was identified using the Digenome provided at github.com/chizksh/digenome-toolkit2.

Western blotting

Harvested cells or mouse brain tissues were dissolved in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (Cell Signaling Technology, Beverly, MA, USA) and incubated on ice for 20 min. Cell lysates were centrifuged at 13,000 × g for 15 min at 4 °C. Total protein concentrations in the supernatants were determined using a Bradford protein assay kit (Thermo Fisher Scientific). Proteins (30 μg) were solubilized in NuPAGE® LDS sample buffer (Invitrogen), denatured for 10 min at 90 °C, and loaded into NuPAGE®4–12% Bis-Tris gels and run in 1x NuPAGE® MES SDS running buffer (Invitrogen) at 120 V for 2 h. The resolved proteins were then transferred onto a 0.45 or 0.2 μm InvitrolonTM polyvinylidene difluoride (PVDF; Invitrogen) membrane in 1× NuPAGE® Transfer buffer with 10% (vol/vol) methanol (EMSURE®) using an XCell IITM Blot Module (Invitrogen) on ice for 1 h 20 min. In case of huge size proteins containing wtHTT and mHTT, proteins were subjected to electrophoresis on a NuPAGE®3–8% Tris-Acetate protein gels in 1x Tris-Acetate SDS Running buffer (Invitrogen) and the membranes transferred in 1x Transfer buffer overnight in the cold room (4 °C). The membranes were blocked with 5% bovine serum albumin (BSA) in 1x TBST [Tris-buffered saline (10 Mm Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.05% Tween 20] at room temperature for 1 h and washed three times with 1x TBST. Blocked membranes were incubated at 4 °C overnight with 5% BSA in 1x TBST and the appropriate primary antibodies listed in Supplementary Table 2. The blots were then washed three times with 1x TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. After washing three times with 1x TBST, immunoreactive bands developed with West-Q Pico ECL Solution (GeneDEPOT, Barker, TX, USA) using the Image Quant LAS-4000 digital imaging system (GE Healthcare, Buckinghamshire, UK). The intensities of the bands were quantified using Multi-Gauge version 3.0 (Fujifilm, Tokyo, Japan). All uncropped Western blot images are presented in Supplementary Fig. 8.

Immunohistochemistry

Animals were euthanized, and slowly perfused with 0.9% normal saline and thereafter the same procedure with 4% paraformaldehyde (PFA). The harvested brain tissues were freezed in frozen section compound (Leica, Wetzlar, Germany) using isopentane and cryosectioned at 16-μm thickness using cryomicrotome (Cryostat Leica 1860, Leica biosystem, MI, Italy). The immunohistochemistry staining was performed on three or four sections over a range of 128 µm. The sections were stained with primary antibodies reported in Supplementary Table 2, as well as secondary antibodies such as Alexa Fluor® 594 goat anti-Rabbit of anti-Mouse (1:400), and Alexa Fluor® 488 goat anti-Rabbit or anti-Mouse (1:400, Invitrogen). The sections were mounted on glass slides with a fluorescent mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI; Vectashield, Vector, Burlingame, CA, USA). The stained sections were analyzed using confocal microscopy (LSM700, Zeiss, Gottingen, Germany) and the density was evaluated using ZEN Imaging Software (Blue edition, Zeiss).

TUNEL assays

For analysis of apoptosis, mouse brain sections were labeled for apoptotic cells by a Fluorometric TUNEL staining according to the manufacturers’ protocols and the selected areas of the striatum were analyzed in four field per tissue. The stained slides containing the cells, and mouse brain sections were analyzed using confocal microscopy (LSM700, Zeiss), and the number of TUNEL+ cells were counted in the fields and the density of TUNEL+ cells in mouse brain sections was evaluated using ZEN Imaging Software (Blue edition, Zeiss).

Statistics and reproducibility

All data were expressed as means ± S.E.M. The variables were compared between groups by an independent t-test and among groups by a one-way analysis of variance (ANOVA) with a post hoc Bonferroni or Tukey comparison using SPSS software (IBM Corporation, Armonk, NY, USA; version 25.0). A P-value <0.05 was considered statistically significant. In vitro experiments were performed at least three times to confirm reproducibility. Both female and male were used in this study and randomly assigned to the in vivo experimental groups. Mice samples were analyzed by molecular study such as qRT-PCR and western blot using data from three independent experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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