Journal: bioRxiv
Article Title: A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation
doi: 10.1101/371500
Figure Lengend Snippet: ( a ) Illustration of the CRISPRa dual vector approach expressing either the single guide RNA (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 days in vitro 4-5 (DIV 4-5). On DIV 11, neurons underwent either immunocytochemistry (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 4,6-diamidino-2-phenylindole (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 ) and ( g-i ). All data are expressed as mean ± s.e.m. Individual comparisons, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.
Article Snippet: A guide scaffold (a gift from Charles Gersbach, Addgene #47108) was inserted into a lentivirus compatible backbone, and EF1a-mCherry was inserted for live-cell visualization.
Techniques: Plasmid Preparation, Expressing, Construct, Transfection, Transduction, In Vitro, Generated, Immunocytochemistry, RNA Extraction, Quantitative RT-PCR, Amplification, Staining
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