Tissue- and time-directed electroporation of CAS9 protein-gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration

Abstract

A rapid method for temporally and spatially controlled CRISPR-mediated gene knockout in vertebrates will be an important tool to screen for genes involved in complex biological phenomena like regeneration. Here we show that in vivo injection of CAS9 protein-guide RNA (gRNA) complexes into the spinal cord lumen of the axolotl and subsequent electroporation leads to comprehensive knockout of Sox2 gene expression in SOX2⁺ neural stem cells with corresponding functional phenotypes from the gene knockout. This is particularly surprising considering the known prevalence of RNase activity in cerebral spinal fluid, which apparently the CAS9 protein protects against. The penetrance/efficiency of gene knockout in the protein-based system is far higher than corresponding electroporation of plasmid-based CRISPR systems. We further show that simultaneous delivery of CAS9-gRNA complexes directed against Sox2 and GFP yields efficient knockout of both genes in GFP-reporter animals. Finally, we show that this method can also be applied to other tissues such as skin and limb mesenchyme. This efficient delivery method opens up the possibility for rapid in vivo genetic screens during axolotl regeneration and can in principle be applied to other vertebrate tissue systems.

Figure 1

Knockout of GFP in the axolotl spinal cord NSCs through CRISPR plasmid electroporation. (a) A scheme of the axolotl spinal cord electroporation procedure. (b) Structure of CRISPR plasmids, pCAGGS-Cas9 (top) and CMV-dsRed-RGR-gRNA (bottom) used for electroporation. HDV, hepatitis delta virus ribozyme; HH, hammerhead ribozyme; NLS, nuclear localisation signal; pA, polyadenylation signal. (c) Fluorescence images of dsRed (red), GFP (green), immunofluorescence for SOX2 (white) combined with DAPI (blue) on 10-μm cryosections showing the loss of GFP expression in a subpopulation of dsRed-labelled spinal cord cells (arrows) at 20 days post electroporation of pCAGGS-Cas9 and CMV-dsRed-RGR-GFP-gRNA plasmids (pCAGGS-Cas9+RGR-GFP-gRNA, upper panel) compared with control (only RGR-GFP-gRNA plasmid, lower panel). Dotted lines define the spinal cord area. Scale bar, 100 μm. (d and e) Quantification of the percentage of (d) GFP⁺ cells over total electroporated dsRed⁺ SOX2⁺ spinal cord cells and (e) GFP⁺ cells over total spinal cord SOX2⁺ NSCs. Loss of GFP expression is observed in ~60% of NSCs that electroporated with the pCAGGS-Cas9 and CMV-dsRed-RGR-GFP-gRNA plasmids (Cas9+RGR-GFP-gRNA) compared with control (Ctr, only CMV-dsRed-RGR-GFP-gRNA plasmid), but in only ~20% cells over total spinal cord SOX2⁺ NSCs. Data are collected from three CRISPR plasmids-electroporated animals and three controls (four to five cross-cryosections per animals).

Figure 2

Knockout of GFP in the axolotl spinal cord NSCs through CAS9–gRNA complex electroporation. (a) DIC (upper panel) and GFP fluorescence (middle panel) images of 6-day regenerates from CAGGS-GFP transgenic axolotls injected with CAS9&GFP–gRNA (left panel) or control (CAS9&Tyr-gRNA, right panel) into the spinal cord lumen and then electroporation. The regenerating spinal cord area, depicted by rectangles, is shown at a higher magnification in the lower panel. Note the clear loss of GFP expression in the regenerating spinal cord tube treated with CAS9&GFP–gRNA as compared with the control. In the CAGGS-GFP transgenic axolotls, GFP is expressed at a much higher level in the mature muscle than in other tissues or in the newly formed immature myofibers. Dotted lines indicate the amputation planes; arrows indicate the spinal cord; dpA, day post amputation. Scale bar, 1 mm. (b) Quantification of the length of 6-day-regenerated spinal cord (solid shapes) and tails (empty shapes) electroporated with CAS9&GFP–gRNA (triangles, n=20) or control CAS9&Tyr-gRNA (Ctr, diamonds, n=12). Error bars, s.d. (c) GFP fluorescence (green) and immunofluorescence for SOX2 (red) combined with DAPI (blue) on 10-μm cross-cryosections show the massive loss of GFP expression in NSCs along the spinal cord from mature ('M') to regenerated ('R') regions of 6-day tail regenerates treated with CAS9&GFP–gRNA compared with the control. The spinal cord areas (squares) are showed at higher magnification as single channel or merged images. Scale bar, 200 μm. (d and e) Quantification of (d) the number of nuclei per cross-section in the tubular spinal cord and (e) the percentage of GFP⁺ SOX2⁺ cells over total SOX2⁺ spinal cord cells of 6-day regenerates at different positions along their length from axolotls treated with CAS9&GFP–gRNA (triangles, n=5) or CAS9&Tyr-gRNA control (diamonds, n=5). Each scale on the x-axis represents ~800 μm of length. M, mature uninjured tail region anterior to the amputation plane; R, regenerate posterior to the amputation plane. Error bars, s.d.; ***P<0.001.

Figure 3

Knockout of Sox2 in the axolotl spinal cord NSCs through CAS9–gRNA complex electroporation. (a) Immunofluorescence for SOX2 (red) and TUJ1 (green) combined with DAPI (blue) on cross-sections show a massive loss of SOX2 immunoactivity in NSCs adjacent to the amputation plane when electroporated with CAS9&Sox2–gRNA (upper panel) compared with CAS9&Tyr-gRNA control (lower panel) at 15 days post electroporation (dpE). The spinal cord areas (squares) are shown at higher magnification as single channel or merged images. Note that SOX2 (labelling NSCs) and TUJ1 (labelling neurons) expression are mutually exclusive in the control spinal cord (lower panel). Arrows indicate the SOX2⁺ cells in the lateral line. Scale bar, 100 μm. (b) Quantification of the percentage of SOX2⁺ cells over total non-neuronal (TUJ1⁻) spinal cord cells adjacent to the amputation plane from axolotls electroporated with CAS9&Sox2–gRNA (circles, n=21) or CAS9&Tyr-gRNA control (diamonds, n=21). Error bars, s.d.; ***P<0.001. (c) Bright field (BF) images of 6-day regenerates. Left: CAS9&Sox2–gRNA; right: CAS9&Tyr-gRNA control. Lower panels show higher magnification images of regenerating spinal cord area (rectangles). In CAS9&Sox2–gRNA-treated axolotls (left lower panel), the spinal cord tube (arrows) that extends into the blastema is significantly shorter and not so clear compared with the control (right lower panel). Dashed lines indicate the amputation planes. Scale bar, 1 mm. (d) Quantification of the length of 6-day-regenerated spinal cord (solid shapes) and tails (empty shapes) electroporated with CAS9&Sox2–gRNA (circles, n=22) or CAS9&Tyr-gRNA control (Ctr, diamonds, n=12). The control data set is same as in Figure 1c. Note that although there is no significant difference of the overall length of the tail, the length of regenerated spinal cord reduced significantly when treated with CAS9&Sox2–gRNA compared with the control. Error bars, s.d.; ***P<0.001. (e) Immunofluorescence for SOX2 (red) and GFAP (green) combined with DAPI on cross-sections along the spinal cord from mature ('M') to regenerated ('R') region of 6-day tail regenerates electroporated with CAS9&Sox2–gRNA (upper panel) or CAS9&Tyr-gRNA control (lower panel). Right lane is the lower-magnification image showing an overview of the tail structure. Note that in the tail blastema, knockout of Sox2 causes reduced cell number in the spinal cord of the CAS9&Sox2–gRNA-treated animals compared with the control. Towards the end of the regenerate ('R2') in the CAS9&Sox2–gRNA-treated animals, GFAP⁺ spinal cord cells are intermingled within the mesenchyme cells, instead of organising into a tubular structure as in the control. Scale bars, 100 μm. (f) Quantification of the number of nuclei per cross-section in the tubular spinal cord of the 6-day regenerate at different positions along its length from axolotls treated with CAS9&Sox2–gRNA (triangles, n=5) or CAS9&Tyr-gRNA control (diamonds, n=5). Each scale on the x-axis represents ~800 μm. M, mature uninjured tail region anterior to the amputation plane; R, regenerate posterior to the amputation plane. Error bars, s.d.; **P<0.005, ***P<0.001.

Figure 4

GFP and Sox2 double-gene knockout in the axolotl spinal cord NSCs through CAS9–gRNA complex electroporation. (a) GFP fluorescence (green) and immunofluorescence for SOX2 (red) combined with DAPI (blue) on cross-sections show a massive loss of GFP and SOX2 expression in NSCs in the mature ('M') and regenerated ('R') region of 10-day tail blastema treated with CAS9&GFP/Sox2–gRNAs (upper panel) compared with the CAS9&Tyr-gRNA control (lower panel). Dotted lines define the spinal cord area; arrows indicate dorsal root ganglia (DRG). Note that the comprehensive loss of GFP and SOX2 occurs only in the spinal cord area but not in the DRG or elsewhere in the tail of CAS9&GFP/Sox2–gRNAs-treated samples compared with the control. CA, cartilage; NT, notochord. Scale bar, 100 μm. (b and c) Quantification of the percentage of SOX2⁺ (b) and GFP⁺ (c) cells over total non-neuronal spinal cord cells at 15 days post electroporation of CAS9&GFP/Sox2–gRNA (circles in b, triangles in c, n=6) or CAS9&Tyr-gRNA control (diamonds, n=6). Neurons in the spinal cord are determined by their location and by the shape of the DAPI-stained nuclei as described in the Materials and methods. Error bars, s.d.; ***P<0.001. (d) Graph shows that the majority (94%) of the targeted NSCs lose both GFP and SOX2 expression (GFP⁻ SOX2⁻) within in the cell population harbouring at least one gene deletion. Only very minority (6%) shows either GFP (GFP⁻ SOX2⁺) or Sox2 (GFP⁺ SOX2⁻) single gene knockout.

Figure 5

GFP knockout in other cell types in the axolotl through CAS9–gRNA complex electroporation. (a and a′) GFP fluorescence (green), PAX7 immunofluorescence (red), DAPI (blue) and DIC images of cross-sections from 21-day regenerating limb treated with CAS9&GFP–gRNAs (a) and CAS9&Tyr-gRNA (control, a′). Boxed areas (number 1–4 in a and a′) are shown at higher magnification in b and b′. Scale bar, 200 μm. (b and b′) Images show the loss of GFP expression in a subpopulation of satellite cells identified by PAX7 staining (first row, arrows), as well as the cells at bone region (second row, dotted line area), dermal mesenchyme (third row, arrows) and epidermis (fourth row, dotted line area), which were identified based on the cell morphology and location, in the limbs treated with CAS9&GFP–gRNAs (b) compared with the control (b′). Red lines indicate the location of basal lamina that separates the epidermis (Epi) from the Dermis (Der).