Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System

Figures

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  Figure 1.. Generation of Single Vector CRISPR/SaCas9 A) Design of AAV-FLEX-SaCas9-U6-sgRNA. (B) Design of AAV-FLEX(1LoxP)-SaCas9-U6-sgRNA. (C) Schematic for designing, generating, and analyzing CRISPR AAVs. (D) Illustration of viral injections into the VTA and guide sequences for Th, Gt(Rosa26)Sor, and Th(h). Exon 2 of Th containing the sequence of the guide is denoted; note that lowercase is intron sequence and uppercase is exon. The intronic sequence containing the guide for Gt(Rosa26)Sor is also denoted, and PAMs are underlined. (E) Example images of coronal VTA sections co-stained for EGFP (for identifying virally transduced and Cre-positive neurons using AAV1-FLEX-EGFP-KASH) and tyrosine hydroxylase (TH; a marker for dopamine neurons) showing loss of TH staining in the 1loxP and FLEX strategies compared with controls. (F) Example images of coronal VTA sections co-stained for GFP and TH in Thlox/lox mice injected with AAV1-CAG-Cre-GFP. Scale bars: 250 mm. (G) Cell count quantification of VTA TH-positive cells (n = 3 mice/group; one-way ANOVA F[4, 10] = 214.3, p < 0.0001; Tukey’s multiple comparisons test, ***p < 0.001). (H) Comparison of knockout efficiencies among the FLEX, 1LoxP, and…

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  Figure 2.. Analysis of Cell Type-Specific Mutagenesis (A) Schematic for isolation, FACS, WGA, PCR, and sequencing (seq) for identifying indels at targeted sites. (B) Schematic of FACS illustrating designated gates (left) and example FACS (right). (C and D) Indel frequencies for the combined gate (C) and bright gates (D) for targeted analysis of Th. (E and F) Indel frequencies for the combined gate (E) and bright gates (F) for targeted analysis of Gt(Rosa26)Sor. (G and H) Sanger sequencing of FACS sorted GFP-negative (top) and GFP-positive (middle) nuclei of mice co-injected with AAV1-FLEX-SaCas9-U6-sgTh (G) and AAV1-FLEX-EGFP-KASH or AAV1-FLEX-SaCas9-U6-sgGt(Rosa26)Sor (H) and AAV1-FLEX-EGFP-KASH into the VTA. Most common indels (bottom) for Th-targeted (G) and Gt(Rosa26)Sor-targeted (H) nuclei. (I and J) Percentage of sequence reads with wild-type, insertion, deletion, or base changes for Th-targeted (I) and Gt(Rosa26)Sor-targeted (J) nuclei.

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  Figure 3.. Alleviating Confounds of Conventional Genetics (A) Design of AAV1-FLEX-SaCas9-U6-sgKcnn3. Exon 1 of Kcnn3 is denoted with guide sequence highlighted in red; the PAM is underlined. (B) Illustration of viral injections into the VTA. (C) Example traces of tail currents evoked following a 500 ms step from ‒70 to 0 mV. (D) Quantification of tail current amplitudes following genetic knockout or CRISPR knockout of Kcnn3 or application of apamin (wild-type, n = 10; Het, n = 19; cKO, n = 15; control, n = 11; sgKcnn3, n = 15; apamin, n = 6; one-way ANOVA F[5, 70] = 12.05, p < 0.0001; Bonferroni’s select comparison, *p < 0.05 and ***p < 0.001). (E) Example traces of action potential firing from control or sgKcnn3-targeted dopamine neurons. (F and G) Quantification of the coefficient of variation of the inter-spike interval (F) and firing rate (G) in control or sgKcnn3-targeted dopamine neurons (control, n = 6; sgKcnn3, n = 9; Student’s unpaired t test, **p < 0.01). For sgKcnn3 control, mice were injected with AAV1-FLEX-SaCas9-sgTh(h). Data are presented as mean ± SEM.

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  Figure 4.. Targeting Difficult-to-Isolate Cell Types (A) Design of AAV1-FLEX-SaCas9-U6-sgGabrg2 and control virus AAV1-FLEX-SaCas9-U6-sgGabrg2TTT. Exon 1 of Gabrg2 is denoted with guide sequence highlighted in red; the PAM is underlined; note that the guide targets the 3′ sequence, so it is written in the reverse complement. (B) Illustration of viral injections into the VTA. (C and D) Representative traces and quantification of mIPSC frequency recorded from VTA (C) dopamine (n = 12 cells/group) or (D) GABA (controls, n = 14 cells; sgGabrg2, n = 16 cells) cells. Left: average mIPSC frequency (Student’s t test, **p < 0.01 and ***p < 0.001). Right: cumulative distribution of inter-event intervals. (E–G) Comparison of locomotion among Vgat-Cre (n = 13), DAT-Cre (n = 13), and control (n = 44) mice (E) across three consecutive days and nights in 15 min time bins, (F) the sum of total distance traveled across 2 consecutive days (one-way ANOVA F[2, 66] = 4.179, p < 0.05; Tukey’s multiple-comparisons test, *p < 0.05), and (G) the sum of total distance traveled across three consecutive nights (one-way ANOVA, F[2, 66] = 5.363, p < 0.001; Tukey’s multiple-comparisons test, *p < 0.05 and **p < 0.01).…

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  Figure 5.. Intersectional Strategy Targeting Slc6a3 Produces Robust DAT Knockout and Hyperactivity (A) Design of AAV1-FLEX-SaCas9-U6-sgSlc6a3 and control virus AAV1-FLEX-SaCas9-U6-sgSlc6a3TTT. Exon 2 of Slc6a3 is denoted with guide sequence highlighted in red; the PAM is underlined. (B) Illustration of viral injections into the VTA. (C) Illustration of intersectional strategy with injection of CAV2-CMV-Cre into the terminals of VTA dopamine neurons in the nucleus accumbens (NAc) and AAV1-FLEX-SaCas9-U6-sgSlc6a3 into VTA dopamine cell bodies in wild-type mice. (D) Example IHC images of DAT and TH in the NAc, dorsal striatum (DStr), and VTA in control, VTA-targeted, or intersect-strategy mice. Scale bar: 250 mm. (E) Western blots from NAc punches probing for DAT and actin comparing the single AAV1-FLEX-SaCas9-U6-sgSlc6a3 VTA injection and the intersect strategy with controls. (F) Quantification of DAT levels from the western blots (controls, n = 7; VTA sgSlc6a3, n = 4; VTA-NAc sgSlc6a3, n = 3; one-way ANOVA, F[2, 11] = 9.055, p < 0.001; Tukey’s multiple-comparisons test, *p < 0.05 and **p < 0.01). (G and H) Locomotion measured for AAV1-FLEX-SaCas9-U6-sgSlc6a3 single VTA injection (controls, n…

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  Figure 6.. Targeted Mutagenesis of Vesicular Glutamate and GABA Transporters (A) Schematic of Cre-dependent AAVs for targeting Vglut1 (Slc17a7), Vglut2 (Slc17a6), and Vgat (Slc32a1). Exons of targeted loci are denoted with guide sequence highlighted in red; the PAM is underlined; note that the Slc32a1 guide targets the 3′ sequence, so it is written in the reverse complement. (B) Schematic of experimental design for testing efficacy of sgSlc17a7 in Vglut1-Cre mice. (C) Example light-evoked EPSC in CM neuron from control and AAV1-FLEX-SaCas9-U6-sgSlc17a7-injected mice. (D) Quantification of light-evoked EPSCs (control n = 12; sgSlc17a7 n = 13; Student’s unpaired t test, ***p < 0.001). (E) Schematic of experimental design for testing efficacy of sgSlc17a6 in Vglut2-Cre mice. (F) Example light-evoked EPSC in VTA neuron from control and AAV1-FLEX-SaCas9-U6-sgSlc17a6 mice. (G) Quantification of light-evoked EPSCs (control n = 11; sgSlc17a6 n = 15; Student’s unpaired t test, ***p < 0.001). (H) Schematic of experimental design for testing efficacy of sgSlc32a1 in Vgat-Cre mice. (I) Example light-evoked IPSC in GABA neuron of the VTA from control and AAV1-FLEX-SaCas9-U6-sgSlc32a1 mice. (J)…

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  Figure 7.. Flp-Dependent CRISPR/SaCas9 Produces Robust Gene Mutagenesis (A) Design of AAV1-FLEXfrt-SaCas9-U6-sgGrin1 and control virus AAV1-FLEXfrt-SaCas9-U6-sgGrin1ATG. Exon 1 of Grin1 is denoted with guide sequence highlighted in red; the PAM is underlined. (B) Illustration of sagittal section with viral injections into the VTA. (C) Representative traces of NMDA and AMPA currents evoked in dopamine neurons by electrical stimulation in WT mice or mice expressing sgGrin1 or sgGrin1ATG. AMPA current recordings were made in the presence of AP5; NMDA currents were determined by subtracting the AMPA current from the compound current trace recorded in ACSF. (D) Quantification of the peak NMDA current as a percentage of the peak AMPA current (n = 10 cells/group; one-way ANOVA, F[2, 27] = 15.55, p < 0.0001; Tukey’s multiple-comparisons test, ***p < 0.001). (E) Sanger sequencing of FACS sorted GFP-positive nuclei from the combined gate of mice co-injected with AAV1-FLEXfrt-SaCas9-U6-sgGrin1 and AAV1-FLEXfrt-EGFP-KASH into the VTA of Th-Flp mice and the most common indels (bottom). (F) Percentage of targeted deep sequencing reads with wild-type, insertion, deletion, or base changes. Data are…