Materials and Methods

Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

MATERIALS AND METHODSAnimalsAdult male Sprague-Dawley rats (230 to 250 g at the start of experiments) were provided by the Department of Laboratory Animal Sciences, Peking University Health Science Center. Rats were maintained in a 12-hour light/12-hour dark cycle with a maximum of four animals per cage and with ad libitum access to food and water. All experimental procedures complied with the guidelines of the Animal Care and Use Committee of the Peking University Health Science Center. Rats were handled for at least 3 days before any experiments were conducted. By the end of the experiment, animals were dissected to perform immunofluorescence staining analysis to verify virus infection, and animals with inappropriate virus infection were excluded from the analysis.gRNA design and AAV productionFor SaCas9 target selection and generation of single gRNA, the 21-nucleotide target sequences were selected to precede a 5′-NNGRRT PAM sequence. The gRNAs were designed using the CRISPR RGEN Tools (www.rgenome.net/cas-designer/) to minimize the off-target effect. gRNAs used in this study are listed in table S1. AAV used in vivo were packaged and purchased from Vigene Biosciences (Jinan, China) or OBiO Technology (Shanghai, China). AAV vectors used in this study are listed in table S2.Cell culture and lentivirus transfectionF98 and C6 rat glioblastoma cell lines and human embryonic kidney cell line 293T were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (HyClone) in a humidified atmosphere with 5% CO2 and 95% air at 37°C.Lentiviral particles were generated by cotransfecting 293T cells with virus packaging vectors (pMD2.G and psPAX2). Transfection was performed using VigoFect (Vigorous Biotechnology Beijing Co. Ltd.) according to the manufacturer’s protocols. Six hours after transfection, the medium was changed. Virus supernatant was harvested 36 and 60 hours after transfection, filtered with a 0.45-μm polyvinylidene difluoride (PVDF) filter (Millipore), ultracentrifuged at 25,000 rpm using a P28S rotor (Hitachi Ltd., Japan), and stocked in a final volume of 100 μl. The titer of the lentivirus used in all cell culture experiments was at least 5.0 × 108 infectious units/ml. F98 and C6 cells were infected with lentivirus at 50 to 60% confluency, followed by selection in DMEM containing puromycin (1 or 6 μg/ml) for 2 to 5 days.Immunoblot analysisCells were collected and lysed in radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktail (Roche Life Science) on ice for 30 min. Cell lysates were clarified by centrifugation at 4°C for 20 min. Total protein concentration was measured by Coomassie Protein Assay Kit (Pierce Chemical, Dallas, TX). Equal amounts of protein in each sample were separated on 8 or 10% SDS–polyacrylamide gel electrophoresis gels and then electrotransferred to PVDF membranes (Millipore). After blocking in 5% nonfat milk for 1 hour at room temperature, the membranes were incubated overnight at 4°C with specified primary antibody against CBP (7389S, Cell Signaling Technology), histone H3 (ab1791, Abcam), Ac-H3K14 (ab52946, Abcam), flag (F1804, Sigma-Aldrich), p300 (sc-48343, Santa Cruz Biotechnology), and β-actin (M177-3, MBL), respectively. After washing three times with tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with the horseradish peroxidase–conjugated goat anti-rabbit/mouse immunoglobulin G antibody (111-035-003/115-035-003, Jackson ImmunoResearch) at room temperature for 1 hour. Protein bands were detected using Western blotting luminol reagent (sc-2048, Santa Cruz Biotechnology). Data were analyzed with the ImageJ software.T7 endonuclease assays for genome modificationGenomic DNA was extracted 3 days after lentivirus transfection using the Universal Genomic DNA Kit (CW2298S, CWBIO) following the manufacturer’s protocol. The genomic region flanking the target sites for each guide sequence was amplified with polymerase chain reaction (PCR) by Q5 Hot Start High-Fidelity 2× Master Mix (E3321S, New England Biolabs) with the following program: preheat at 98°C for 30 s, 35 cycles of three-step amplification (98°C for 5 s, 62°C for 10 s, and 72°C for 20 s), and final extension at 72°C for 2 min. The primers used are listed in table S3. A total of 200 ng of the purified PCR products was mixed with buffer 2 and ultrapure water to a final volume of 19 μl. Hybridization reactions were performed with the following program: 95°C for 5 min, 95° to 85°C at −2°C/s, and 85° to 25°C at −0.1°C/s. Then, 1 μl of EnGen T7 Endonuclease I was added, and the mixture was incubated at 37°C for 15 min. One microliter of Proteinase K was added to stop the reaction followed by gel electrophoresis on a 2% agarose gel.Stereotactic injection of AAVThe rat was anesthetized by intraperitoneal injection of 1% pentobarbital sodium (0.1 g/kg, intraperitoneally) and positioned in a stereotactic instrument equipped with a heating pad (RWD Life Science, Shenzhen, China). The scalp was shaved, a small incision was made along the midline to expose the skull, and a small hole was drilled in the skull above the requisite injection site. A total of 0.5 μl of AAV was injected into the dCA1 anteroposterior (AP): −3.6 mm; mediolateral (ML): ±2.2 mm; dorsoventral (DV): −2.8 mm, PL (AP: 2.9 mm; ML: ±0.5 mm; DV: −3.0 mm), IL (AP: 2.8 mm; ML: ±0.5 mm; DV: −4.5 mm), or amygdala (AP: −2.8 mm; ML: ±4.8 mm; DV: −8.4 mm) at a flow rate of 0.1 μl/min via a microinjection pump. After each injection, the needle was held in place for 5 min to allow for virus diffusion and gradually withdrawn to prevent possible leakage from the needle track. The incision was sutured, and rat was returned to its home cage for 1-week recovery before subsequent experiments.ImmunofluorescenceRats were anesthetized with 1% pentobarbital sodium and intracardially perfused with 4% paraformaldehyde. Brains were postfixed with 4% paraformaldehyde for 12 hours and kept in 20 and 30% sucrose solutions in turn for dehydration. Thirty-micrometer sections were sliced coronally using a cryostat microtome (model 1950, Leica). Free-floating sections were washed in phosphate-buffered saline, blocked with a buffer containing 5% bull serum albumin and 0.3% Triton X-100 for 1 hour, incubated with primary antibodies at 4°C overnight, washed three times in phosphate-buffered saline, and then incubated with secondary antibody solution containing 4′,6-diamidino-2-phenylindole for 1 hour at 37°C. The second antibodies used for the staining were Alexa Fluor 488/594/647 goat anti-rabbit/mouse immunoglobulin G (ZSGB-BIO, Beijing). Sections were lastly washed three times and mounted on microscope slides using the mounting medium (ZSGB-BIO, Beijing). Images were captured under a Leica TCS SP8 confocal laser-scanning microscope, and colocalization analysis and merged images were processed according to our previous work (25).ElectrophysiologyRats were anesthetized with isoflurane and perfused transcardially with chilled sucrose-based cutting solution: 234 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgSO4, and 10 mM glucose (pH 7.4; 335 to 340 mosmol). The brain was removed quickly, and coronal bilateral slices that contained the PL were cut at 350 μm with a vibratome (VT1000S, Leica). These slices were incubated in 37°C artificial cerebral spinal fluid solution (aCSF) consisting of 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 2 mM CaCl2, 2 mM MgCl2, and 10 mM glucose (305 to 310 mosmol), aerated with 95% O2 and 5% CO2 to a final pH of 7.4. After 1-hour incubation, slices were transferred carefully to the recording chamber by super-fusing aCSF at room temperature.A slice was viewed with an upright microscope (Axioskop Fsmot, Zeiss) equipped with infrared differential interference contrast optics. Pyramidal cells of the PL were recognized via a 40× water immersion lens. The recording pipettes (3 to 5 megohms) were filled with solution that contained 140 mM K-gluconate, 8 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 10 mM Hepes, 2 mM Mg–adenosine triphosphate, and 0.3 mM Na–guanosine triphosphate (pH 7.2; 290 to 320 mosmol). Voltage and current signals were recorded from PL neurons using an Axon 200B amplifier. The spike firing pattern of the AP was used to electrophysiologically identify pyramidal neurons, which exhibited significant spike frequency adaptation in response to a depolarization current. The APs were recorded using the current-clamp mode.Contextual fear conditioningFor CFC, we used a chamber (25 cm wide by 25 cm long by 25 cm high) with distinct visual cues and a grid floor, which consisted of 36 stainless steel rods (fear conditioning and startle system, Panlab, Spain). The chamber was cleaned with 70% ethanol before the introduction of each rat. Rats were kept in the conditioning chamber for a total of 300 s, when foot shocks (2 s, 0.75 mA) were delivered at 120, 180, and 240 s. During the test, rats were placed into the same context and allowed to explore for 300 s to monitor their freezing behavior. Freezing was defined as being motionless and measured by Packwin V2.0 software (Panlab, Spain). The amount of time spent freezing was expressed as a percentage of total session time. For context-specific fear conditioning, chamber A had a black plastic wall and roof, while chamber B was white.For fear extinction, rats were placed in the conditioning chamber for a total of 300 s, when foot shocks (2 s, 0.50 mA) were delivered at 120, 180, and 240 s. Two weeks later, the rats were taken on Dox (100 mg/kg) for 24 hours to open a window of activity-dependent labeling. Rats were exposed to the context for 300 s for cell labeling, and Dox diets were withdrawn immediately after context exposure. One week later, rats were placed into the conditioned context and allowed to explore for 300 s for three consecutive days to monitor their fear extinction.Fluorescence-activated cell sortingFour weeks after delivery of AAV9-hSyn-SaCas9-3×flag and AAV9-U6-gRNA-mix-CMV-GFP into the PL, the rat was anesthetized with 1% pentobarbital sodium, and the brain was quickly removed and sectioned on a vibratome on ice. Individual slices of interest were transferred to a small dish containing cold (4°C) dissection media: 116 mM NaCl, 5.4 mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 1.5 mM CaCl2, 1 mM MgSO4, 0.5 mM EDTA, 25 mM glucose, and 1 mM cysteine, bubbled with 95% O2 and 5% CO2. The PL was dissected and treated with dissection media with papain (1 mg/ml; {"type":"entrez-nucleotide","attrs":{"text":"LS003119","term_id":"1321651605","term_text":"LS003119"}}LS003119, Worthington) for 30 min at 37°C. The tissue pieces were dissociated into single cells by gentle trituration and filtered through a 70-μm cell strainer (F613462, BBI). Sorting was performed by a fluorescence-activated cell sorter (BD Biosciences) in the single-cell sorting mode to select neurons with high enhanced green fluorescent protein fluorescence for subsequent deep sequencing or RNA sequencing (RNA-seq) analysis.Deep sequencing for off-target analysisPotential off-target sites were identified using CRISPR RGEN Tools (www.rgenome.net/cas-offinder/). For the three CBP targets described above, we computationally selected 33 candidate off-target sites in the rat reference genome (rn5) that were followed by a 5′-NNGRRT PAM with less than five mismatched bases. The genomic region flanking the target sites for each guide sequence was PCR amplified with the following protocol: preheat at 98°C for 30 s, 35 cycles of three-step amplification (98°C for 5 s, 62°C for 10 s, and 72°C for 20 s), and final extension at 72°C for 2 min. The primers for each potential off-target site are listed in table S4. PCR products were purified using DNA Clean-up Kit (CW2301, CWBIO) following the manufacturer’s recommended protocol and sequenced with the MGISEQ-200 (BGI, China). Reads were filtered by an average Phred quality (Q score) > 20 and perfect sequence matches to amplicon forward and reverse primers. Reads from on- and off-target loci were analyzed by first performing alignments against amplicon sequences that included 25 nucleotides upstream and downstream of the target site (a total of 77 bp). Alignments, meanwhile, were analyzed for indels from 5 bp upstream to 5 bp downstream of the target site (37 bp), and indels in this region were discarded with no change among six nucleotides upstream of the PAM sequence (cutting site by SaCas9).RNA-seq analysisTotal RNA of the neurons by FACS was extracted by TRIzol reagent and quantified with the ND-2000 (NanoDrop Technologies). The libraries were sequenced by BGISEQ-500 (igeneCode Biotech, Beijing), and qualified reads were mapped to the rat reference genome (rn5). The expression level for each transcript was calculated using FPKM (fragments per kilobase of transcript per million fragments mapped). Hierarchical clustering and Gene Ontology were used to analyze differentially expressed genes, with log2 fold change >2 and false discovery rate <0.01 as significantly enriched.Statistical analysisThe data were expressed as means ± SEM and analyzed using GraphPad Prism. Statistical analysis of two experimental groups was performed using two-tailed Student’s t tests, and comparisons of two groups with different time points were performed using two-way analysis of variance (ANOVA), with P < 0.05 as statistically significant.

Article TitleDevelopment of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

Abstract

A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)–associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system–based technique, and its combined application with anterograde/retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection- and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down thecbp(CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection- and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection- and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies.


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