Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9

Abstract

CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca²⁺ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.

Fig. 1

CRISPR-Cas9 Transfection experimental design and Protocol. a. General experimental timeline of CRISPR-Cas9 transfection in hDRG culture and each consequent experimental verification steps b. Visual representation of transfection reagent and CRISPR plasmid formulation; a 1:1 mixture of DNA dilution and lipofectamine dilution incubated for 15 min before added to the hDRG cultured cells and incubated for 24 h.c. Measurement of neuronal yield per one hDRG dissociated across 3 different age groups. Each dot represents the neuronal yield for a single donor. d. Plasmid maps for CRISPR-Cas9 gene editing, one containing an mCherry reported tag and another containing a GFP reporter tag. Plasmid map images were obtained from GeneCopoeia website.

Fig. 2

Validation of CRISPR-Cas9 transfection with reporter tag and T7 endonuclease assay. a. MTS % change in viability in hDRG cultured cells treated for either 24- or 48 - hours with the TRPV1 gRNA plasmid (500ng) demonstrating cells should not be exposed to the lipid-plasmid complex for more than 24-hours. There were no statistically significant differences between any of the groups using a one-way ANOVA using Tukey’s multiple comparison test b. Fluorescent live images of mCherry reporter tag expression in the CRISPR-Cas9 TRPV1 KO plasmid treated cells (bottom), compared to the negative control cells (top). Representative brightfield images (left) and the fluorescent images (right) were taken using a Biorad ZOE Fluorescent cell imager. Scale bar 100 μm c. % transfection efficiency for the TRPV1 CRISPR plasmid quantified by fluorescent tag expression in neurons alone (77%) and in the entire hDRG culture (63%). Each point represents the average of at least 100 neuronal and at least 600 non-neuronal cells quantified from 3 individual replicates. The transfection efficiency denoted as all cells includes both neuronal and non-neuronal cells. d. Visual workflow of T7 endonuclease I assay used to confirm CRISPR-Cas9 genetic edit. e. Representative image of DNA gel demonstrating multiple DNA band fragments in the TRPV1 CRISPR Plasmid exposed sample compared to the single negative control sample band (original gel in supplementary Fig. 2). Quantification of the bands was done through ImageJ to obtain the fraction of cleaved PCR products and the % of gene edit modification.

Fig. 3

Validation of decreased protein expression and protein function in TRPV1 CRISPR cells. hDRG cells were transfected with CRISPR-Cas9 plasmid targeting TRPV1 and were either fixed or lysed for protein 5-days later and analyzed via western blot or immunocytochemistry. (a) Representative 10X fluorescent images of TRPV1 in NC or CRISPR-Cas9 treated cells. Peripherin (blue) marked neurons, mCherry was seen only in the CRISPR-treated cells, and TRPV1 is significantly decreased these cells. The merge images of all three channels are shown for each negative control and CRISPR treated cells with the corresponding 20X image. (b) Data for the mean fluorescent intensity of TRPV1 (Green) was quantified as means ± SEM of n = 10 neurons per group in 3 individual replicates normalized to NC control average (100%). ****P < 0.0001 by unpaired two-tailed t-test. Scale bars = 100 μm (c) The representative Western blot and quantification of TRPV1; data was normalized to total protein, then further normalized to the negative control group (100%) (original blot in supplementary Fig. 2). ** = p < 0.01 by unpaired two-tailed t-test. (d) 72-hours post-transfection, the cells were incubated with Tyrode’s buffer containing 2 μm Fura 2-AM/ Pluronic F-127 covered at room temperature for an hour then incubated with Tyrode’s buffer supplemented with 2.5 mM probenecid for 20–30 min. A 2-minute baseline was recorded followed by 800nM capsaicin stimulation recorded for 2-minutes in 30 s intervals. A decreased capsaicin evoked calcium response is seen in the TRPV1-CRISPR-KO cells compared to the NC capsaicin cells. The data was reported as the 340/380 ratio and normalized to a 4-point negative control baseline. NC-Capsaicin vs. NC vehicle same time point; **** = p < 0.0001; ** = p < 0.01; * = p < 0.05;2-Way ANOVA with Tukey’s post hoc test. Data was measured in two separate donors with 4–6 individual replicates per group. (e) Representative area under the curve (AUC) comparing vehicle, NC (Cap 800nM), and TRPV1 KO (800nM) capsaicin response post 120 s timepoint; data normalized to NC (cap 800nM) (100%). **** = p < 0.0001; *** = p < 0.001; ** = p < 0.01 by ordinary one-way ANOVA with Tukey’s multiple comparison test.

Fig. 4

CRISPR-Cas9 genetic editing of NTSR2 and CACNA1E in hDRG cultures. a. Representative fluorescent live cell images of GFP reporter tag expression in the CRISPR-Cas9 CACNA1E KO plasmid treated cells (bottom), compared to the negative control cells (top). Representative brightfield images (left) and the fluorescent images (right) were taken using a Biorad ZOE Fluorescent cell imager. Scale bar 100 μm. b. Percent transfection efficiency for the CACNA1E CRISPR plasmid quantified by fluorescent tag expression in neurons alone and in the entire hDRG culture. c. Percent transfection efficiency for the NTSR2 CRISPR plasmid quantified by fluorescent tag expression in neurons alone and in the entire hDRG culture. (Images not shown). Each point represents the average of at least 100 neuronal and at least 600 non-neuronal cells quantified from 3-individual replicates. The transfection efficiency denoted as all cells includes both neuronal and non-neuronal cells. d. CellTiter Glo 2.0 Assay demonstrating viability of cells may differ between the cells exposed to plasmids containing the GFP compared to the mCherry reporter. Both NTSR2 and CACNA1E DNA plasmids with GFP reporter tags show a significant decrease in viability compared to non-treated control cells but not in comparison to lipofectamine treated NC cells. Both lipofectamine alone and the mCherry tag plasmid (TRPV1) show no significant decrease in viability compared to the non-treated control cells. Luminescence was measured across two different donors with 3 individual replicates per treatment group using a Tecan Spark 20 plate reader; *** = p < 0.001; ** = p < 0.01; by 1-Way ANOVA with Tukey’s post-hoc test e. Representative image of DNA gel demonstrating multiple DNA band fragments in the NTSR2 & CACNA1E CRISPR plasmid exposed sample compared to the single negative control sample band. f. Representative Western blot and quantification of NTSR2; data was normalized to GAPDH, then normalized to the negative control group (100%). ** = p < 0.01; * = p < 0.1 by unpaired 2-tailed t-test. g. Representative Western blot and quantification of Cav2.3; data was normalized to GAPDH, then normalized to the negative control group (100%). * = p < 0.05 by unpaired 2-tailed t-test. (original gel and blots in supplementary Fig. 2).

Fig. 5

Immunocytochemistry validation of CRISPR editing of NTSR2 and Cav2.3 protein levels in hDRG culture. Representative 10X fluorescent images staining for NTSR2 and Cav2.3 of negative control or CRISPR-Cas9 treated hDRG cultures. (a) Peripherin (blue) was used as the neuronal marker, the reporter tag (GFP) was seen only in the CRISPR treated cells, while the target of interest is significantly decreased in the perspective CRISPR treated cells. The merge images of all three channels are shown for each negative control and CRISPR treated cells with the corresponding 20X image. (b) Data for the mean fluorescent intensity for the targets of interest Cav2.3 (Red), and NTSR2 (Red) was quantified as means ± SEM of n = 10 neurons per group in 3 individual replicates; data normalized to NC control (100%).****P < 0.0001 by unpaired two-tailed t-test. Scale bars = 100 μm.