Journal: eNeuro

Article Title: A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

doi: 10.1523/ENEURO.0495-18.2019

Figure Lengend Snippet: CRISPRa gene induction in HEK293T cells, C6 cells, and primary rat neurons under ubiquitous and neuron-selective promoters. A , Illustration of the CRISPRa dual vector approach expressing either the sgRNA or the dCas9-VPR construct driven by EF1α, PGK, CAG, or SYN promoters. B , dCas9-VPR co-transfected with sgRNAs targeted to the human FOS gene results in induction of FOS mRNA in HEK293T cells regardless of the promoter driving dCas9-VPR ( n = 6, unpaired t test; EF1α t (5.308) = 8.034, p = 0.0004; PGK t (5.138) = 5.943, p = 0.0018; CAG t (6.097) = 11.15, p < 0.0001; SYN t (5.064) = 4.67, p = 0.0053). C , dCas9-VPR co-nucleofected with sgRNAs targeting the rat Fos gene induces Fos mRNA in a C6 glioblastoma cell line ( n = 6, unpaired t test; EF1α t (5.006) = 8.699, p = 0.0003; PGK t (5.067) = 6.640, p = 0.0011; CAG t (5.148) = 18.32, p < 0.0001; SYN t (5.000) = 8.631, p = 0.0003). D , Lentiviral transduction of primary rat cortical neurons reveals that only dCas9-VPR driven by the SYN promoter results in induction of Fos mRNA ( n = 6, unpaired t test; EF1α t (6.912) = 0.492, p = 0.6378; PGK t (9.491) = 0.710, p = 0.4950; SYN t (5.234) = 7.593, p = 0.0005). E , Experimental timeline for in vitro CRISPRa in neurons. Primary rat neuronal cultures are generated and transduced with dual sgRNA/dCas9-VPR lentiviruses at DIV4–DIV5. On DIV11, neurons underwent either ICC to validate viral expression or RNA extraction followed by RT-qPCR to examine gene expression. F , ICC reveals high co-transduction efficiency of guide RNA (co-expressing mCherry, signal not amplified) and dCas9-VPR (FLAG-tagged) lentiviruses in primary neuronal cultures. Cell nuclei are stained with DAPI. Scale bar = 50 μm. G–I , dCas9-VPR increases gene expression for a panel of genes in cortical, hippocampal, or striatal cultures. Data are expressed as fold change of the target gene’s expression relative to dCas9-VPR targeted to a non-targeting control (bacterial LacZ gene; n = 4–6, unpaired t test; cortical: Reln t (5.438) = 12.590, p < 0.0001; Nr4a1 t (3.250) = 5.692, p = 0.0086; Egr1 t (5.084) = 6.233, p = 0.0015; Fos t (5.571) = 16.770, p < 0.0001; Fosb t (5.167) = 19.570, p < 0.0001; hippocampal: Nr4a1 t (5.760) = 7.140, p = 0.0005; Reln t (6.102) = 7.236, p = 0.0003; Egr1 t (5.091) = 8.565, p = 0.0003; Fos t (6.668) = 27.410, p < 0.0001; Fosb t (5.021) = 12.210, p < 0.0001; striatal: Ascl1 t (5.111) = 9.383, p = 0.0002; Reln t (5.667) = 12.790, p < 0.0001; Egr1 t (5.760) = 10.320, p < 0.0001; Isl1 t (5.047) = 6.074, p = 0.0017; Ebf1 t (5.012) = 7.007, p = 0.0009; Fos t (5.026) = 5.349, P 0.003; Fosb t (4.015) = 5.057, p = 0.0071). dCas9-VPR with a sgRNA targeted to the bacterial LacZ gene is used as a non-targeting control in panels B–D , G–I . All data are expressed as mean ± SEM. Individual comparisons; ** p < 0.01, *** p < 0.001, **** p < 0.0001. Transgene expression and proviral integration in primary neurons are shown in Extended Data . CRISPR sgRNA and RT-qPCR primer sequences are provided in Extended .

Article Snippet: To validate expression of the dCas9-VPR cassette, anti-FLAG primary antibody (1:5000 in PBS with 10% Thermo Blocker BSA and 1% goat serum, Thermo Fisher Scientific catalog #MA1-91878, RRID: AB_1957945 ) was incubated overnight at 4°C.

Techniques: Plasmid Preparation, Expressing, Construct, Transfection, Transduction, In Vitro, Generated, RNA Extraction, Quantitative RT-PCR, Gene Expression, Amplification, Staining, Control, CRISPR

Journal: eNeuro

Article Title: A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

doi: 10.1523/ENEURO.0495-18.2019

Figure Lengend Snippet: CRISPRa-mediated induction of Fosb in hippocampal, striatal, and cortical neurons in vivo . A–C , Lentiviral infusions were bilaterally targeted to the brain region of interest in adult male rats ( n = 4 rats/region). Two weeks following stereotaxic viral infusions, animals were transcardially perfused and IHC was performed to measure Fosb upregulation. IHC reveals high transduction efficiency of the guide RNA (expressing mCherry, signal not amplified) bilaterally in ( A ) the CA1 region of the dorsal hippocampus, ( B ) the nucleus accumbens core (NAc), and ( C ) the medial PFC. Fosb protein is enhanced in the hemisphere that was infused with the Fosb -targeting sgRNA (right) compared to the hemisphere that received a sgRNA targeting the bacterial LacZ gene (left). Cell nuclei were stained with DAPI. Scale bar = 500 μm. Schematics of target regions are adapted from Paxinos and Watson. D–F , dCas9-VPR increases the number of Fosb+ cells in the CA1, NAc, and PFC, compared to a non-targeting control ( LacZ ; n = 4, ratio paired t test; CA1: t (3) = 8.73, p = 0.003, R 2 = 0.96; NAc: t (3) = 4.62, p = 0.019, R 2 = 0.87; PFC: t (3) = 3.43, p = 0.041, R 2 = 0.79). All data are expressed as mean ± SEM. Individual comparisons; * p < 0.05 and ** p < 0.01. Or: oriens layer, Py: pyramidal cell layer, Rad: radiatum layer, LMol: lacunosum moleculare, DG: dentate gyrus, ac: anterior commissure, LV: lateral ventricle.

Article Snippet: To validate expression of the dCas9-VPR cassette, anti-FLAG primary antibody (1:5000 in PBS with 10% Thermo Blocker BSA and 1% goat serum, Thermo Fisher Scientific catalog #MA1-91878, RRID: AB_1957945 ) was incubated overnight at 4°C.

Techniques: In Vivo, Transduction, Expressing, Amplification, Staining, Control