Research ArticleNEUROSCIENCE

Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

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Science Advances  18 Mar 2020:
Vol. 6, no. 12, eaay6687
DOI: 10.1126/sciadv.aay6687
  • Fig. 1 SaCas9 inactivates the cbp in vitro with high efficiency.

    (A) A schematic of CMV-SaCas9-P2A-mCherry-U6-gRNA lentivirus vector. SaCas9 was labeled with a flag epitope tag. mCherry was cleaved from SaCas9 through the P2A self-cleaving peptide, indicating the expression of vector. (B) Graphical representation of the rat cbp gene locus showing gRNA target sites, with three gRNAs spacing less than 200 bp apart in exon 2. (C and D) Expression of SaCas9 was evaluated by immunofluorescence (C) and immunoblot (D) 3 days after lentivirus transfection in rat glioma cells. Scale bar, 20 μm. WT, wild type. (E) Western blot analysis showing decreased CBP expression in F98 cells 1 week after lentivirus transfection. (F) Western blot analysis showing decreased CBP expression and acetylation of H3K14 in cells expressing SaCas9 and multiple gRNA-1+4+5 (gRNA-mix). (G) T7 endonuclease assay for Cas9-mediated cleavage in F98 cells transfected with SaCas9 and indicated gRNA, with red triangles indicating cleavage fragments. (H and I) Immunofluorescence staining of CBP (H) and Ac-H3K14 (I) 1 week after CRISPR-SaCas9 targeting of cbp locus in F98 cells, with white arrows indicating decreased CBP expression (H) and acetylation of H3K14 (I) in SaCas9+ and gRNA+ cells. Scale bars, 10 μm.

  • Fig. 2 Perturbing cbp in postsynaptic PL neurons receiving dCA1 inputs.

    (A) A schematic of the experiment: AAV1-CaMKII-Cre-P2A-GFP injection in bilateral dCA1 and AAV9-hSyn-DIO-mCherry injection in bilateral PL. (B) Coronal sections showing dCA1 labeled with GFP and PL labeled with Cre-dependent mCherry and dCA1 axons expressing GFP. Scale bars, 200 μm. (C) A schematic of the experiment: rAAV2-retro-hSyn-Cre injection in bilateral PL and AAV9-hSyn-DIO-mCherry injection in bilateral dCA1. (D) Coronal sections showing dCA1 labeled with Cre-dependent mCherry. Scale bar, 200 μm. (E) A schematic of the experiment: AAV1-CaMKII-Cre was injected bilaterally into the dCA1, and a mixture (1:1 ratio) of AAV9-hSyn-DIO-SaCas9 and AAV9-U6-gRNA-mix-CMV-GFP was injected into bilateral PL. (F) Expression of SaCas9 and gRNA-GFP in the PL 4 weeks after AAV delivery. Scale bar, 50 μm. (G) Cotransfection efficiency of gRNA and SaCas9 (n = 4 slices from three animals). (H) Immunofluorescence staining of CBP (top) and Ac-H3K14 (bottom) in the PL 4 weeks after AAV injection. The white arrows indicate decreased CBP expression and H3K14 acetylation in SaCas9- and gRNA-GFP+ neurons. Scale bars, 10 μm. (I) Quantification of CBP- or Ac-H3K14+ cells in the targeted neuronal population (gRNA+ + SaCas9+) (n = 5 slices from three animals). (J) Immunofluorescence staining of PSD-95 in the PL 4 weeks after AAV injection, with the white arrow indicating decreased PSD-95 expression in gRNA-GFP+ neurons. Scale bar, 10 μm. (K) Quantification of PSD-95+ cells in the targeted neuronal population (gRNA+) (n = 4 slices from three animals). (L) Decreased dendritic spines in PL neurons after cbp knockdown by SaCas9 and gRNA-mix. Control neurons were transfected with the same AAV with gRNA-mix replaced with gRNA-control. Scale bar, 5 μm. (M) Spine density quantification (t test, t12 = 4.88, **P < 0.01; n = 3 animals per group, two to three slices per animal). (N) An example of AP responses to positive current steps recorded from PL neurons of control and cbp knockdown groups. (O) Number of APs induced by injected currents in PL neurons from control and cbp knockdown rats [n = 10 and 15 neurons; **P < 0.01, two-way analysis of variance (ANOVA)]. Group effect: F1,72 = 11.38, P < 0.01; current intensity effect: F7,112 = 18.95, P < 0.01; interaction: F7,72 = 0.84, P > 0.05.

  • Fig. 3 Perturbing cbp in PL engram cells impairs remote memory.

    (A) A schematic of the experiment: A mixture (1:1 ratio) of AAV9–c-fos–rtTA-U6-gRNA-mix and AAV9-TRE3G-SaCas9 was injected into bilateral PL. With Dox, training induced the expression of rtTA, which bound to TRE, drove the expression of SaCas9, and induced indel generation in the cbp locus of activated PL cells. (B) Scheme of behavioral test. Rats were trained in the context for fear conditioning 3 weeks after AAV injection. Two weeks later, the rats were given Dox for 1 day and then exposed to the context for engram cell labeling. Recall test was performed 1 week later. (C) Immunofluorescence staining of SaCas9 in the PL 2 days after context exposure. Left (Dox −, shock +): with shock during CFC training and without Dox during context exposure; middle (Dox +, shock +): with shock during CFC training and with Dox during context exposure; right (Dox +, shock −): without shock during CFC training and with Dox during context exposure. Scale bar, 50 μm. (D) Immunofluorescence staining of c-Fos in PL, amygdala, and dHP 2 hours after the CFC test. Scale bars, 200 μm. (E) c-Fos+ cell counts in PL, amygdala, and dHP following the CFC test (n = 5 per group, one-way ANOVA. PL: F2,12 = 107.90, P < 0.01. “Dox −, shock +” versus “Dox +, shock +”, **P < 0.01. “Dox +, shock +” versus “Dox +, shock −”, **P < 0.01; amygdala: F2,12 = 81.45, P < 0.01. “Dox −, shock +” versus “Dox +, shock +”, **P < 0.01. “Dox +, shock +” versus “Dox +, shock −”, P > 0.05; dHP: F2,12 = 0.01, P > 0.05). (F) Rats showed less freezing during the recall test after cbp knockdown (n = 8 per group, one-way ANOVA, F2,21 = 16.64, P < 0.01. “Dox −, shock +” versus “Dox +, shock +”, *P < 0.05. “Dox +, shock +” versus “Dox +, shock −”, *P < 0.05). (G) A schematic of the experiment: AAV9–c-fos–rtTA and AAV9-TRE3G-SaCas9 were injected in the PL. With Dox, training induced the expression of rtTA, which bound to TRE and labeled the activated cells with SaCas9. There was no indel generation in the cbp locus due to the absence of gRNA, ensuring the normal function of neurons. (H) Representative coronal section of the PL labeled with SaCas9 and c-Fos 2 hours after the CFC test. SaCas9 and c-Fos double-positive neurons were indicated by arrows. Scale bar, 20 μm. (I) Scheme of behavioral test. Three weeks after AAV injection as shown in (A), the rats were fear-conditioned in contexts A and B. Two weeks later, the rats were exposed to context A, returned to home cage, and taken on Dox for 1 day before exposure to context B. The recall test for contexts A and B was performed 1 week later. (J) Rats showed less freezing during the CFC test in context B than in context A (t test, n = 8 per group. Context exposure: t14 = 0.20, P > 0.05; CFC test: t14 = 2.63, *P < 0.05).

  • Fig. 4 PL engram neurons receiving monosynaptic inputs from dCA1 contribute to remote memory.

    (A) Schematic of the experiment: AAV1-hSyn-Cre-P2A-GFP was injected bilaterally into the dCA1, and a mixture (1:1 ratio) of AAV9-c-fos-rtTA-U6-gRNA-mix and AAV9-TRE3G-DIO-SaCas9 was injected into bilateral PL. (B) Scheme of behavioral test. Rats were trained in the context for fear conditioning 3 weeks after AAV injection. Two weeks later, the rats were given Dox for 1 day and exposed to the context for cell labeling. Recall test was performed 1 week later. (C) Coronal sections showing dCA1 labeled with GFP and PL labeled with Cre- and activity-dependent SaCas9 and dCA1 axons expressing GFP. Scale bars, 200 μm (left) and 50 μm (right). (D) SaCas9+ cell counts in the PL 2 days after context exposure (n = 6 slices from three animals). (E) cbp knockdown in PL engram cells with direct dCA1 inputs decreased the duration of freezing in the probe test (t test, t16 = 2.31, *P < 0.05; n = 9 per group). Rats that were never exposed to Dox served as controls.

  • Fig. 5 Perturbing cbp in presynaptic IL extinction-ensemble cells via the CRISPR-SaCas9 system impairs extinction learning.

    (A) A schematic of the experiment: A mixture (1:1 ratio) of AAV9–c-fos–rtTA-U6-gRNA-mix and AAV9-TRE3G-SaCas9 was injected into bilateral IL. (B) Scheme of behavioral testing. Rats were trained in the context for fear conditioning 3 weeks after AAV injection. Two weeks later, the rats were given Dox for 1 day, followed by exposure to the context for labeling IL extinction-ensemble cells. Rats were reexposed to the context for three consecutive days for extinction test. (C) Immunofluorescence staining of c-Fos in the IL after context exposure. Scale bars, 200 μm. (D) c-Fos+ cell counts in the IL following context exposure (t test, t8 = 8.18, **P < 0.01; n = 5 slices from three animals in either group). (E) Averaged freezing time during the recall test for cbp knockdown and control groups (two-way ANOVA, **P < 0.01; n = 8 per group. Group effect: F1,28 = 19.45, P < 0.01; time effect: F3,28 = 42.97, P < 0.01; interaction: F3,28 = 0.76, P > 0.05). Rats not exposed to Dox served as control. (F) Graph depicting the percentage of c-Fos+ + mCherry+/mCherry+ (left) and c-Fos+ + mCherry+/c-Fos+ (right) neurons after context exposure (n = 5 slices from three animals). mCherry+: amygdala-projecting IL neurons; c-Fos+: activated neurons. Amygdala-projecting IL neurons (14%) were activated (c-Fos+ + mCherry+/mCherry+), and 12% of the activated neurons projected to amygdala (c-Fos+ + mCherry+/c-Fos+). (G) Illustration of amygdala-projecting IL neurons (mCherry+) that showed immunolabeling of c-Fos protein 2 hours after context exposure. Scale bars, 100 μm (IL) and 200 μm (Amy). (H) Schematic of the experiment: rAAV2-retro-hSyn-Cre-P2A-GFP was injected bilaterally into the amygdala, and a mixture (1:1 ratio) of AAV9–c-fos–rtTA-U6-gRNA-mix and AAV9-TRE3G-DIO-SaCas9 was injected into bilateral IL. (I) Coronal sections showing some amygdala-projecting IL neurons (GFP+) colabeled with Cre- and activity-dependent SaCas9. Scale bar, 100 μm. (J) Cotransfection efficiency of GFP and SaCas9 (n = 5 slices from three animals). Dependent expression of SaCas9 on Cre-GFP resulted in 90% SaCas9+ + GFP+/SaCas9+ neurons. (K) Averaged freezing time during extinction test for cbp knockdown and control groups (two-way ANOVA, **P < 0.01; n = 8 per group. Group effect: F1,28 = 10.64, P < 0.01; time effect: F3,28 = 88.17, P < 0.01; interaction: F3,28 = 0.14, P > 0.05). Rats that were never exposed to Dox served as control.

  • Fig. 6 CRISPR-SaCas9 has superior targeting specificity in vivo.

    (A) A flowchart showing purification of GFP+ neurons from the rat brain 4 weeks after AAV9-hSyn-SaCas9 and AAV9-U6-gRNA-mix-CMV-GFP injection in the PL. (B) Western blot analysis showing highly enriched SaCas9 within sorted GFP+ neurons. (C) Cleavage levels at putative genomic off-target loci containing fewer than five mismatches (white cells) for cbp targets 1, 4, and 5 were analyzed by deep sequencing. (D) Graphical representation of the rat cbp locus showing gRNA target location. Targeted genomic locus is marked as blue. PAM sequence is marked as red. The red arrowheads indicate CRISPR-SaCas9 cutting site. Representative mutation patterns detected by sequencing of cbp locus are shown below. Top: wild-type sequence; blue dashes: deleted bases; blue bases: insertion or mutations (indel). (E) Hierarchical clustering of differentially expressed genes (log2 fold change >2 and false discovery rate <0.01 as significant difference) detected by RNA sequencing (RNA-seq) 4 weeks after AAV delivery. Relative log [FPKM (fragments per kilobase of transcript per million fragments mapped)] expression levels of genes are displayed in red-blue color scale. (F) The differentially expressed genes from control and cbp knockdown groups were classified by Gene Ontology analysis.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaay6687/DC1

    Fig. S1. SaCas9 inactivates the p300 gene in vitro with high efficiency.

    Fig. S2. Neuronal specificity for spine density analysis.

    Fig. S3. Amygdala-projecting IL neurons are activated during extinction.

    Table S1. gRNA sequences targeting cbp gene or p300 gene.

    Table S2. AAV vectors used.

    Table S3. Primer sequences for T7 endonuclease assays.

    Table S4. Primer sequences for off-target analysis.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. SaCas9 inactivates the p300 gene in vitro with high efficiency.
    • Fig. S2. Neuronal specificity for spine density analysis.
    • Fig. S3. Amygdala-projecting IL neurons are activated during extinction.
    • Table S1. gRNA sequences targeting cbp gene or p300 gene.
    • Table S2. AAV vectors used.
    • Table S3. Primer sequences for T7 endonuclease assays.
    • Table S4. Primer sequences for off-target analysis.

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