Materials and methods

A Cre-dependent CRISPR/dCas9 system for gene expression regulation in neurons

Cultured neuron experiments

Primary rat neuronal cultures were generated from embryonic day 18 rat striatal tissue as described previously (Savell et al., 2019a). Briefly, cell culture wells were coated overnight at 37° C with poly-L-lysine (0.05 mg/ml for culture wells supplemented with up to 0.05 mg/ml Laminin) and rinsed with diH2O. Dissected tissues were incubated with papain for 25 min at 37°C. After rinsing in Hank’s Balanced Salt Solution (HBSS), a single cell suspension of the tissue was re-suspended in Neurobasal media (Invitrogen) by trituration through a series of large to small fire-polished Pasteur pipets. Primary neuronal cells were passed through a 100 μM cell strainer, spun and re-suspended in fresh media. Cells were then counted and plated to a density of 125,000 cells per well on 24-well culture plate with or without glass coverslips (60,000 cells/cm). Cells were grown in Neurobasal media plus B-27 and L-glutamine supplement (complete Neurobasal media) for 11 DIV in a humidified CO2 (5%) incubator at 37° C.

For viral transduction, cells were transduced with lentiviruses on DIV 4 or 5. All viruses had a minimum titer of 1×109 GC/ml, with a target multiplicity of infection (MOIs) of at least 1000. After an 8-16 hr incubation period, virus-containing media was replaced with conditioned media to minimize toxicity. A regular half-media change followed on DIV 8. On DIV 11, transduced cells were imaged and virus expression was verified prior to RNA extraction. EGFP and mCherry expression was also used to visualize successful transduction using a Nikon TiS inverted epifluorescence microscope.

RNA extraction and RT-qPCR

Total RNA was extracted (RNAeasy kit, Qiagen) with DNase treatment (RNase free DNAse, Qiagen), and reverse-transcribed (iScript cDNA Synthesis Kit, Bio-Rad). cDNA was subject to qPCR for genes of interest, as described previously (Savell et al., 2016). A list of PCR primer sequences is provided in Supplementary Data Table 1.

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Supplemental Data Table 1.

Sequences of primers and sgRNAs

CRISPR-dCas9 construct design

To achieve transcriptional activation or inactivation, lentivirus-compatible plasmids were engineered to express dCas9 fused to either VPR or KRAB-MeCP2, based on existing published plasmids (Addgene plasmid # 114196 (Savell et al., 2019a); Addgene plasmid # 155365 (Duke et al., 2020)). A Cre-dependent DIO version of the dCas9-VPR construct was generated by insertion of LoxP and Lox2272 sequences flanking the dCas9-VPR cassette. dCas9-VPR was PCR-amplified to insert additional restriction sites (KpnI, BmtI, and BspDI) to allow for LoxP and Lox2272 insertion and was subsequently inserted in reverse orientation to create an intermediate construct via sequential digest and ligation (AgeI, EcoRI). LoxP and Lox2272 sites were amplified from a DIO construct (Addgene plasmid # 113685 (Don et al., 2017)) to create restriction sites (KpnI and BmtI around one set; BspDI and KpnI around another set) and were inserted into the intermediate construct via sequential restriction digest and ligation (KpnI, BmtI, BspDI and EcoRI). Intron-containing dCas9-VPR was constructed by the insertion of a gBlock containing the SV40 intron into the original dCas9-VPR plasmid via Gibson assembly (Gibson Assembly kit, New England BioLabs). The SVI-FLEX construct was built via Gibson assembly of the original dCas9-VPR backbone and two gBlocks encoding the SV40 intron sequence, LoxP and Lox2272 sites (sequences based on DIO construct described above) and dCas9-part1 in inverted orientation. The intron-containing CRISPRi construct (SVI-dCas9-KRAB-MeCP2) was built via restriction digest and Gibson assembly (SfiI, EcoRI, and Xhol, Gibson Assembly kit, New England BioLabs) of the KRAB-MeCP2 and SVI-dCas9-VPR constructs. A Cre-encoding construct (Addgene plasmid # 49056 ((Kaspar et al., 2002)) was used to amplify and insert a Cre transgene into a lentivirus compatible backbone that contained the hSYN promoter and expressed mCherry for visualization via Gibson assembly. To create an additional Cre construct, mCherry was replaced with GFP via sequential digest and ligation (EcoRI and XhoI). dCas9-VPR-expressing constructs were co-transduced with sgRNA-containing constructs. Gene-specific sgRNAs were designed using an online sgRNA tool, provided by the Zhang Lab at MIT (crispr.mit. edu) and inserted in a previously described lentivirus compatible sgRNA scaffold construct (Addgene plasmid # 114199 (Savell et al., 2019b)). To ensure specificity, all CRISPR crRNA sequences were analyzed with the National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool (BLAST) and Cas-OFFinder (http://www.rgenome.net/cas-offinder/). sgRNAs were designed to target GRM2, Tent5b, Sstr2, Gadd45b, and Fos respectively. A list of the target sequences is provided in Supplementary Data Table 1. crRNA sequences were annealed and ligated into the sgRNA scaffold using the BbsI or BsmBI cut site. Plasmids were sequence-verified with Sanger sequencing; final crRNA insertion was verified using PCR. Lentivirus-compatible SYN-SVI-DIO-dCas9-VPR and SYN-SVI-DIO-dCas9-KRAB-MeCP2 plasmids will be made available on Addgene (plasmid #164576 and # 170378).

Lentivirus production

Viruses were produced in a sterile environment subject to BSL-2 safety by transfecting HEK293T cells with specified CRISPR-dCas9 plasmids, the psPAX2 packaging plasmid, and the pCMV-VSV-G envelope plasmid (Addgene plasmids #12260 & #8454) with FuGene HD (Promega) for 40-48 hrs as previously described (Savell et al., 2019b). Viruses were purified using filter (0.45 μm) and ultracentrifugation (25,000 rpm, 1 hr 45 min) steps. Viral titer was determined using a qPCR Lentivirus Titration Kit (Lenti-X, qRT-PCR Titration Kit, Takara). For smaller scale virus preparation, each sgRNA plasmid was transfected in a 12-well culture plate as described above. After 40-48 hr, lentiviruses were concentrated with Lenti-X concentrator (Takara), resuspended in sterile PBS, and used immediately. Viruses were stored in sterile PBS at −80°C in single-use aliquots.

HEK293T cell culturing and transfection

HEK293T cells were obtained from American Type Culture Collection (ATCC catalog# CRL-3216, RRID:CVCL_0063) and cultured in standard HEK Media: DMEM (DMEM High glucose, pyruvate; Gibco 11995081) supplemented with 10% bovine serum (Qualified US Origin; BioFluid 200-500-Q) and 1U Penicillin-Streptomycin (Gibco 15140122). Cells were maintained in T75 or T225 tissue flasks. At each passage, cells were trypsinized for 1-3 min (0.25% trypsin and 1 mM EDTA in PBS pH 7.4) at room temperature. For transfection experiment cells were plated in 24 well plates and transfected with FuGene HD (Promega).

Luciferase assay

Bidirectional regulation by SVI-DIO CRISPRa and CRISPRi machinery was examined using a previously described Fos luciferase reporter plasmid (Duke et al., 2020). 80,000 HEK293T cells were plated in 500 μl HEK Media. After cells reached 40-50% confluence, 500 ng total plasmid DNA was transfected with 1.5 μl FuGene HD (Promega) as follows: 50 ng of luciferase plasmid, 450 ng in 1:2 molar ratio of total sgRNA:CRISPRa or CRISPRi plasmid. A luciferase glow assay was performed according to manufacturer’s instructions 40 hrs following transfection (Thermo Scientific Pierce Firefly Glow Assay; Thermo Scientific 16177). Cells were lysed in 100 μl 1x Luciferase Cell Lysis Buffer while shaking at low speed and protected from light for 30 min. 20 μl of cell lysate was then added to an opaque 96-well microplate (Corning 353296) and combined with 50 μl 1x D-Luciferin Working Solution supplemented with 1x Firefly Signal Enhancer (Thermo Scientific Pierce Firefly Signal Enhancer; Thermo Scientific 16180). Following a 10 min dark incubation period to allow for signal stabilization, luminescence was recorded using a Synergy 2 Multi-Detection Microplate Reader (BioTek). Luminescence in dCas9-KRAB-MeCP2 and dCas9-VPR experiments was recorded with a read height of 1 mm, 1 s integration time, and 135 or 100 ms delay, respectively. Representative images of luciferase reporter activity presented in Figure 4 were captured using an Azure c600 imager (Azure Biosystems).

Article TitleA Cre-dependent CRISPR/dCas9 system for gene expression regulation in neurons

Abstract

Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customizations for different applications. The simplicity and modularity of CRISPR-based systems have transformed this aspect of genome editing, providing a variety of possible applications and targets. However, there are currently few available tools for cell-selective CRISPR regulation in neurons. Here, we designed, validated, and optimized CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems for Cre recombinase-dependent gene regulation. Unexpectedly, CRISPRa systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction in the absence of Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in both HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate highly inducible genes (GRM2, Tent5b, and Fos) as well as moderately inducible genes (Sstr2 and Gadd45b) in a Cre-specific manner. Furthermore, to illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from of a luciferase reporter in HEK293T cells only in the presence of Cre. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and will enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.


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