Journal: bioRxiv

Article Title: Cell-type targeted CRISPR/Cas9 Clock knockdown in mouse VTA dopamine neurons alter sleep, behavior, and cellular excitability

doi: 10.64898/2026.06.03.730017

Figure Lengend Snippet: ( A ) Schematic of the Cre-dependent AAV-CMV-DIO-SaCas9-U6-sgRNA vector. ( B ) sgRNA target site within the Rosa26 locus, showing the sgRNA target sequence (red) and PAM sequence (blue); sgRNA target sequence is anti-sense relative to the gene coding sequence. ( C ) Representative sanger sequencing traces from Cre- and Cre+ samples, with the expected SaCas9 cut site indicated (arrow). ( D ) In vitro validation workflow. NIH/3T3 cells were co-transfected with AAV-CMV-DIO-SaCas9-U6-Rosa26 sgRNA and AAV-Ef1a-mCherry-IRES-Cre. mCherry-positive cells isolated by FACS and analyzed by amplicon sequencing. ( E ) Pie chart showing average indel frequency (wild-type, deletion, insertion), with individual sample editing efficiencies shown below. ( F ) Bar graph showing average distribution of indel sizes, grouped by deletion and insertion length in base pairs. ( G ) In vivo validation workflow. DAT-Cre mice were co-injected with AAV-DIO-KASH-EGFP and AAV-DIO-SaCas9-U6-Rosa26 sgRNA. ( H ) Pie chart showing the average indel frequency, with individual sample editing efficiencies shown below. ( I ) Bar graph showing average distribution of indel sizes, grouped by deletion and insertions (bp).

Article Snippet: Correct insertion of sgRNA sequences was confirmed by Sanger sequencing (Plasmidsaurus).

Techniques: Plasmid Preparation, Sequencing, In Vitro, Biomarker Discovery, Transfection, Isolation, Amplification, In Vivo, Injection

Journal: bioRxiv

Article Title: Cell-type targeted CRISPR/Cas9 Clock knockdown in mouse VTA dopamine neurons alter sleep, behavior, and cellular excitability

doi: 10.64898/2026.06.03.730017

Figure Lengend Snippet: (A) Mouse clock gene structure with sgRNA target sites in Exons 9, 10, and 16; sgRNA (blue) and PAM (red) sequences indicated. sgRNA target sequence is anti-sense relative to the gene coding sequence. Sanger traces from GFP+ nuclei confirm editing at each site; GFP- traces not shown. ( B-D ) Indel distributions for Clock sgRNAs #1-#3 from in vivo virally-transduced nuclei as pie charts and scatter plots of individual editing efficiencies. sgRNA #3 achieved 69% editing efficiency. ( E ) In situ hybridization quantifying Clock mRNA in Cas9-expressing VTA neurons. ( F ) Representative fluorescence images for Cas9 (green), DAT (red) and Clock (cyan). Scale bar = 100 μm. ( G ) Violin plot quantifying Clock mRNA levels in Cas9-expressing cells. Clock-targeted sgRNA significantly reduced mRNA relative to Rosa26 control. ( H ) Experimental design for immunohistochemistry (IHC). Virus expressing Cas9 and sgRNAs targeting Clock (C3 or C2+3) was injected at different titers (low: 5 × 10¹¹, high: 5 × 10¹²). After 4 weeks, brain sections were processed for IHC. (I ) Representative IHC images showing Clock (red), and HA-tag (Cas9+, magenta). Scale bar = 100 μm. ( J ) Clock intensity in HA+ (Cas9+) cells. ( K ) Bar graph showing knockdown efficiency. *p< 0.05, **p<0.01, ****p<0.0001

Article Snippet: Correct insertion of sgRNA sequences was confirmed by Sanger sequencing (Plasmidsaurus).

Techniques: Sequencing, In Vivo, In Situ Hybridization, Expressing, Fluorescence, Control, Immunohistochemistry, Virus, Injection, Knockdown