H11-Cas9 mice on C57BL/6J [B6J.129(Cg)-Igs2tm1.1(CAG-cas9*)Mmw/J; stock #028239; laboratory of M. Winslow, Stanford University, Stanford, CA]  constitutively expressing Streptococcus pyogenes Cas9 (Cas9) were purchased from Jackson Laboratory (Bar Harbor, ME). Homozygous H11-Cas9 mice were crossed to generate animals used in all experiments of this study. Mice within litters were randomized for the treatment groups. Experimenters were blinded to treatment. Animals were euthanized and excluded from tissue processing or further analysis if they exhibited hydrocephalus. All animal use and treatments were approved by the Biogen Institute Animal Care and Use Committee and followed the National Institute of Health Guide for the Care and Use of Laboratory Animals.
The sgRNA design for mouse NeuN (sgNeuN) (5′-GTTTGGGCTGCTGCTTCTCCG-3′) and for LacZ (sgLacZ) (5′-GTGCGAATACGCCCACGCGAT-3′) was previously described (Hana et al. co-submitted for publication). The sgRNA designs for mouse GFAP (sgGFAP), and mouse MOG (sgMOG) were designed in Benchling [26,27,28] based on specificity and efficiency scores. Seven GFAP and seven MOG sgRNA designs were screened in vitro with Cos1 and Neuro-2a cell lines for CRISPR efficiency (Supplementary Figs. 1, 2). The NeuN sgRNA, the most efficient GFAP sgRNA design #6 (5′-GAAGCCAGCATTGAGCGCCC-3′) and the most efficient MOG sgRNA design #4 (5′-GATGACAACTGGAGGAGAAGG-3′) were further used for AAV vector production. All sgRNAs contain a backbone structurally optimized for binding of Cas9 (5′-GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3′) .
Plasmid design, vector production, and purification
GFAP and MOG overexpression vectors contain a CMV promoter that drives expression of mouse GFAP or mouse MOG fused with a Myc epitope tag. The Cas9 expression vector is a gift from Douglas Larigan (pVAX1-FLAG-NLS-SpCas9-NLS) which has a CMV promoter that drives expression of Cas9. Each AAV vector contains a pol III U6 promoter that drives expression of a sgRNA and a CBA promoter that drives expression of a green fluorescence protein (eGFP) fused with a nuclear localization signal (NLS). Single-stranded viral vectors were packaged into either AAV9, AAV-PHP.B, or AAV-PHP.eB at a titer of 5E13 vg/ml and were produced and purified (PackGene Biotech, Worcester, MA). Triple-plasmid transfection using polyethylenimine (PEI, Polyscience) was carried out to produce the recombinant AAV. The transfer plasmids AAV plasmid encoding the cargo described in the previous sgRNA design method section, pRep2CapX of AAV-X encoding Rep2 and CapX proteins plasmids, and pHelper were co-transfected into HEK293T cells. The cells were cultured in Dulbecco’s modified essential medium (DMEM; Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin–streptomycin antibiotics (Gibco, USA) at 37 °C. When the cells reached 80% confluence, they were transfected in 150 mm plates with 12 μg of pHelper plasmid, 10 μg of AAV pRep2CapX plasmids, and 6 μg of transfer AAV plasmids encoding the cargo for each plate. At 72 h post transfection, cells were harvested by 4000 × g centrifugation at 4 °C for 30 min. The pellet was collected and re-suspended in buffer containing 10 mM Tris-HCl, pH 8.0. The suspension was subjected to four freeze–thaw cycles by dry ice/ethanol and a 37 °C water bath. The cell debris was sonicated and then digested with DNase I (200 U in 1.5 ml) for 1 h at 37 °C. Following centrifugation at 10,000 × g for 10 min at 4 °C, the supernatant was collected as the AAV crude lysate. The crude lysate was diluted with 10 mM Tris-HCl pH 8.0 to a final volume of 10 ml and then bottom-loaded to a discontinuous gradient of 15, 25, 40, and 60% Iodixanol in a 39 ml ultracentrifuge tube (QuickSeal, 342414). After ultracentrifugation at 350,000 × g and 18 °C for 1 h, 3 ml fractions at lower position of 40% and 0.5 ml of 60% upper layer were collected. Ultracentrifugation was repeated at 350,000 × g and 18 °C for 1 h one more time and the fractions were desalted using a 100 kDa Cutoff Ultrafiltration tube (15 ml; Millipore, USA). The purified AAV were stored at −80 °C before usage. The viral titers were determined by SYBR Green qPCR.
Neonatal intracerebroventricular (ICV) injections
On postnatal day 0 (P0), neonatal mice were cryo-anesthetized and then injected with AAV-sgNeuN/sgGFAP/sgMOG at either a low (5E10 vg) or high (20E10 vg) dose at a total volume of 4 µL. Mice injected with AAV-sgLacZ were only injected at a high dose. Viruses were diluted with phosphate-buffered saline (PBS) and mixed with Fastgreen dye (final concentration: 0.25%). Mock injected mice received an injection of 4 µL sterile PBS. The ICV injection procedure was adapted from Kim et al. . ICV injections were performed perpendicular to the skull, located 1 mm lateral to the superior sagittal sinus, unilaterally between lambda and bregma, to a depth of 2 mm. Injections were performed with a 33-gauge, 10 µL, 45° bevel Hamilton syringe (Hamilton Company, Reno, NV, USA). Injection efficiency was monitored by the spread of the dye mixture throughout the lateral and the third ventricles. Afterwards, pups were placed inside of a warming chamber to recover body heat. Injected mice were weaned at 4 weeks and the tissues were harvested at 5 weeks for eGFP RNAscope, sgNeuN, and sgGFAP studies. Injected mice were weaned at 5 weeks and the tissues were harvested at 6 weeks for the sgMOG studies.
In situ hybridization (ISH) RNAscope
The whole brains and spinal cords were removed from mice. Samples were fixed in 10% neutral buffer formalin, processed, and embedded into paraffin. Brains were transected sagittally and embedded in the midline down orientation, and spinal cords were transected and embedded in the transverse orientation. ISH staining was performed on a Leica Biosystems’ BOND RX Autostainer (Leica Biosystems) using RNAscope® 2.5 LSx Reagent Kit—RED (Advanced Cell Diagnostics) . Staining was done according to the manufacturer’s instructions. Briefly, 5 µm thick sections were deparaffinized and rehydrated then subjected to target retrieval for 15 min at 95 °C using Leica Epitope Retrieval Buffer 2 (ER2) and protease treatment for 15 min at 40 °C, followed by specific probe-SpCas9 (ACDBio, 475788) hybridized to target RNA for the Cas9 homozygous and WT pups, and probe-eGFP (ACDBio, 400288) for the AAV-PHP.eB and mock transduced pups, respectively. The signal was amplified using a cascade of amplifier hybridizations to binding sites. Fast Red chromogenic detection was then performed. The background staining was assessed by negative control probe-dapB (ACDBio, 312038). Figures 1, 2 are from n = 3, biological replicates for Cas9 versus wild type and eGFP versus mock mice.
Peggy Sue automated western blot
The brain was sub-dissected into the cortex, hippocampus, subcortex, and cerebellum, and half of the spinal cord was collected. Tissues were homogenized in tissue lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X, 1% Na-deoxycholate (Sigma, D6750-106), 0.1% SDS, 8M Urea (Sigma, U4883), 5 mM EDTA, complete protease inhibitor (Roche, 04693124001), PhosSTOP phosphatase inhibitor (Roche, 4906845001), 1 mM phenylmethanesulfonylfluoride fluoride (Sigma, 93482), 1 mM dithiothreitol, 10 mM sodium fluoride, and 1 mM sodium orthovanadate (New England Biosciences, P07585)] by TissureLyser II (Qiagen). Briefly, tissues were placed in Safelock tubes (Eppendorf, 4036-3352) with a 5 mm stainless steel bead (Qiagen, 69989) and were homogenized twice with rapid agitation for 3 min each with a frequency of 30 Hz (TissueLyzer II). Homogenates were subsequently cleared via centrifugation at 4 °C, 375 × g for 20 min and the supernatant was collected. Lysates were electrophoresed through an automated Western Peggy Sue system with the 12–230 kDa detection module according to manufacturer’s instructions (Protein Simple, SM-S001) . Lysates were diluted to the linear dynamic range in 0.1× Sample buffer (Protein Simple, San Jose, CA, 042-195). For NeuN protein analysis, lysates were incubated with 1:250 of anti-NeuN antibody (CST, clone D3S3I, 12943) and either 1:100 anti-GAPDH antibody (CST, clone 14C10, 2118) or 1:500 anti-B-actin (CST, clone 8H10D10, 3700). For GFAP protein analysis, samples were incubated with 1:100 anti-GFAP (CST, clone GA5, 3670) and 1:100 anti-GAPDH. For MOG protein analysis, samples were incubated with 1:100 anti-MOG (CST, clone E5K6T, 96457) and 1:100 anti-GAPDH. Anti-mouse detection module (Protein Simple, DM-002) and anti-rabbit detection module (Protein Simple, DM-001) were used following incubation with corresponding primary antibodies. Figures 3–5 are from n = 2–7, biological replicates for mice injected with low and high doses of AAV9, AAV-PHP.B, and AAV-PHP.eB.
In vitro sgRNA screen in Cos1 cell line
The sgRNAs were compared for their efficiency in facilitating CRISPR-mediated disruption of the target gene expressed from a co-transfected plasmid in Cos1 cells (ATCC, Manassas, VA, CAT#CRL-1650). Cos1 cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin–streptomycin (Gibco, 15070-063), and 2 mM L-glutamine. FuGENE HD (Promega, San Luis Obispo, CA, E2311) was used for transfection according to the manufacturer’s instructions. Briefly, the GFAP or MOG overexpression plasmid, Cas9 expression plasmid, and sgRNA plasmid were mixed in a 1:4:5 ratio to a total of 0.5 µg, and incubated with 1.5 µL of FuGENE HD in 25 µL Opti-MEM (Thermofisher, 31985962) prior to adding to the cultured cells. Twenty-four hours after transfection, cell lysates were harvested in 200 µL Novex Tris-Glycine SDS Sample Buffer (1.6×, Thermofisher, LC2676) with NuPage Sample Reducing Agent (Thermofisher, NP0009) and heated at 70 °C for 5 min (ThermoMixer C, Eppendorf). Lysates were sonicated at a force of 4 for 20 strokes (60 Sonic Dismembrator, Fisher Scientific). The lysates were subjected to western blotting analysis.
In vitro sgRNA screen in Neuro-2a cell line
Neuro-2a cell line with tetracycline-inducible Cas9 expression, (SL508, GeneCopoeia, Rockville, MD), was also used to examine sgRNAs for their efficiency in facilitating CRISPR-mediated disruption of the target gene expressed from a co-transfected plasmid. Neuro-2a cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin–streptomycin (Gibco, 15070-063), and 2 mM L-glutamine. Cells were treated with 0.01, 0.1, or 1 µg/ml of doxycycline to induce expression of Cas9. After 24 h, cells were co-transfected with the GFAP or MOG overexpression plasmid and sgRNA plasmid in a 1:9 ratio by FuGENE (Promega, San Luis Obispo, CA, E2311) according to the manufacturer’s instructions. 24 h after transfection, cell lysates were harvested in 200 µL Novex Tris-Glycine SDS Sample Buffer (1.6×, Thermofisher, LC2676) with NuPage Sample Reducing Agent (Thermofisher, NP0009) and heated at 70 °C for 5 min (ThermoMixer C, Eppendorf). Lysates were sonicated at a force of 4 for 20 strokes (60 Sonic Dismembrator, Fisher Scientific). The lysates were subjected to Western blotting analysis.
Western blotting and antibodies
Lysates were electrophoresed through NuPage 4–12% Bis-Tris Midi Gels (Thermofisher, WG1403BX10) and transferred to nitrocellulose membranes in Trans-blot Turbo Transfer Pack (Bio-Rad, 1704159) using Trans-blot Turbo transfer system (Bio-Rad). The blocking of membranes and subsequent antibody incubations were performed by using Odyssey blocking buffer (LI-COR Biosciences, 927-50000) according to the manufacturer’s instructions. Primary antibodies against Myc (1:2000, Enzo BML-SA294-0500), b-tubulin (1:5000, Li-Cor, 926-422-11), b-actin (1:10,000, Li-Cor, 926-422-10) and FLAG (F1804, Millipore Sigma) were purchased from commercial sources. The IRDye 800CW-conjugated donkey anti-mouse (Li-Cor, 925-32212) and IRDye 680CW-conjugated donkey anti-rabbit (Li-Cor, 925-68073) secondary antibodies were obtained from Li-Cor Biosciences. Immunoblot signals were visualized by the Odyssey CLx infrared imaging and quantified by Li-Cor Odyssey application software.
In Figs. 3–5, quantification was performed for each AAV variant within each CNS region. The averages of the protein signals from AAV-CRISPR/sgLacZ (not shown) were normalized to 100% as baseline (dotted line). Remaining protein levels after CRISPR-mediated protein disruption are presented as means ± standard error of the mean (s.e.m.). Statistical analyses (a two-way ANOVA followed by a Tukey’s test) were performed using GraphPad Prism 7 software. Experimental groups were considered to be significant for p values < 0.05 and <0.01 for all experiments. The p value was only corrected within each CNS region. Protein levels significantly different between AAV variants are denoted with a “*” sign. Within each AAV variant, protein levels significantly different from the corresponding baseline (sgLacZ) are denoted with a “#” sign.