plasmid encoding spcas9  (ToolGen Incorporated)

Journal: bioRxiv

Article Title: CRISPR/CAS9-MEDIATED LOSS OF VESICULAR GLUTAMATE TRANSPORTER IN SEROTONIN NEURONS OF THE DORSAL RAPHE NUCLEUS LEADS TO SYNAPTIC CHANGES AND ANXIETY-LIKE BEHAVIORS

doi: 10.64898/2026.02.23.707446

Figure Lengend Snippet: ( A ) Distribution of 5-HT infected neurons expressing mCherry (red) in the DRN and the MRN following intracerebral injection of AAV placed in the DRN of ePet-cre;Cas9-eGFP mice. ( B ) Orthogonal views of axons (mCherry+, red) arising from 5-HT infected neurons of the DRN showing VGluT3+ (white) axon varicosities in the lateral hypothalamic area of control, VGluT3 conditional KO and VGluT3 overexpressing mice.

Article Snippet: This plasmid containing the 2 gRNAs was co-transfected along with a puromycin-resistant plasmid encoding Streptococcus pyogenes Cas9 (SpCas9) (Addgene, plasmid #62988) in mouse fibroblasts NIH-3T3 (ATCC, CRL-1658) using lipofectamine LTX (Invitrogen).

Techniques: Infection, Expressing, Injection, Control

Journal: bioRxiv

Article Title: CRISPR/CAS9-MEDIATED LOSS OF VESICULAR GLUTAMATE TRANSPORTER IN SEROTONIN NEURONS OF THE DORSAL RAPHE NUCLEUS LEADS TO SYNAPTIC CHANGES AND ANXIETY-LIKE BEHAVIORS

doi: 10.64898/2026.02.23.707446

Figure Lengend Snippet: ( A ) Schematic of the breeding for the new ePet-cre;Cas9-eGFP mouse line. ( B ) Cas9 and eGFP are expressed in 5-HT neurons of the DRN. TpH (purple) and GFP (green) immunopositive neurons were counted in the rostral (bregma –4.36 mm, upper row), mid (bregma –4.60 mm, mid row) and caudal (bregma –4.84mm, lower row) regions of the DRN. Arrows indicate TpH+/GFP- neurons and arrowheads point to TpH+/GFP+ neurons. Cells immunoreactive for both TpH and GFP (green dots) or only TpH (purple dots) in the DRN are depicted in the schematic illustrations in the left column. DRN, dorsal raphe nucleus; DRD, dorsal part of the DRN; DRV, ventral part of the DRN; DRL, lateral part of the DRN; DRI, interfascicular part of the DRN. Scale bar: 100 µm.

Article Snippet: This plasmid containing the 2 gRNAs was co-transfected along with a puromycin-resistant plasmid encoding Streptococcus pyogenes Cas9 (SpCas9) (Addgene, plasmid #62988) in mouse fibroblasts NIH-3T3 (ATCC, CRL-1658) using lipofectamine LTX (Invitrogen).

Techniques:

Journal: bioRxiv

Article Title: CRISPR/CAS9-MEDIATED LOSS OF VESICULAR GLUTAMATE TRANSPORTER IN SEROTONIN NEURONS OF THE DORSAL RAPHE NUCLEUS LEADS TO SYNAPTIC CHANGES AND ANXIETY-LIKE BEHAVIORS

doi: 10.64898/2026.02.23.707446

Figure Lengend Snippet: ( A-B ) Indels generated from the co-transfection of NIH-3T3 cells with the plasmids encoding for gRNAs used in the present study were analyzed using TIDE method for CRISPR/Cas9 gene editing. Indel spectrum and quality control for gRNA1 and gRNA2 are shown in A and B , respectively.

Article Snippet: This plasmid containing the 2 gRNAs was co-transfected along with a puromycin-resistant plasmid encoding Streptococcus pyogenes Cas9 (SpCas9) (Addgene, plasmid #62988) in mouse fibroblasts NIH-3T3 (ATCC, CRL-1658) using lipofectamine LTX (Invitrogen).

Techniques: Generated, Cotransfection, CRISPR, Control

Journal: bioRxiv

Article Title: CRISPR/CAS9-MEDIATED LOSS OF VESICULAR GLUTAMATE TRANSPORTER IN SEROTONIN NEURONS OF THE DORSAL RAPHE NUCLEUS LEADS TO SYNAPTIC CHANGES AND ANXIETY-LIKE BEHAVIORS

doi: 10.64898/2026.02.23.707446

Figure Lengend Snippet: ( A ) Experimental timeline showing viral injection (surgery) followed by behavioral tests used to assess the effect of VGluT3 depletion or overexpression on motricity, anxiety and depressive-like behaviors of ePet-cre;Cas9-eGFP mice. ( B-K ) Violin plots of data distributions (probability density), median and interquartile ranges for the different behavioral tests. Coordination, endurance, and muscular strength as assessed with the rotarod ( B ) were affected in VGluT3 conditional KO mice which fell faster than control ( P = 0.004). No difference in the spontaneous motor activity (distance travelled, vertical and stereotypic activity; C-E ) as assessed in the open field was noted between the three experimental groups. Anxiety-like behaviors were scored in the open field ( F ), nestlet shredding test (NST, G ) and elevated plus maze (EPM, H ). VGluT3 conditional KO mice tend to spend less time in the center of the open field ( P = 0.1) and significantly less time in the open arms of the EPM compared to control mice ( P = 0.005). Nest building activity was significantly increased in VGluT3 conditional KO mice ( P < 0.0001), whereas it was significantly reduced in VGluT3 overexpressing mice ( P = 0.038) when compared to control animals. Assessment of depressive-like behaviors was done using the tail suspension test (TST, I ), social interaction test ( J ) and sucrose preference test ( K ). No depressive-like behavior was observed in either VGluT3-depleted or VGluT3 overexpressing mice. However, VGluT3 conditional KO mice displayed increased social interactions ( P < 0.0005), while VGluT3 overexpressing mice showed a greater preference for sweetened water ( P = 0.023). Dots represent individual values obtained for each mouse. * P < 0.05; ** P < 0.005; *** P < 0.001; **** P < 0.0001 using the two-tailed Mann-Whitney U test.

Article Snippet: This plasmid containing the 2 gRNAs was co-transfected along with a puromycin-resistant plasmid encoding Streptococcus pyogenes Cas9 (SpCas9) (Addgene, plasmid #62988) in mouse fibroblasts NIH-3T3 (ATCC, CRL-1658) using lipofectamine LTX (Invitrogen).

Techniques: Injection, Over Expression, Control, Activity Assay, Suspension, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Non-canonical function of the splicing activator U2AF2 in promoting intron retention in the lncRNAs PURPL and MALAT1

doi: 10.64898/2026.02.19.706780

Figure Lengend Snippet: (A) Schematic of the PURPL IR reporter used in the screen to identify IR regulators. The pLCHKO vector contains the puromycin resistance gene used for selection. The transgene also contains the genomic sequences of exon 2 (161 bp), intron 2 (1,943 bp), and exon 3 (163 bp) of the PURPL gene under a Doxycycline-inducible promoter. A hybrid guide (hg)RNA for guiding SpCas9 and LbCas12a is expressed under a U6 promoter. A construct with a hgRNA targeting an intergenic region was used as a negative control. Top arrow: The hgRNA is cleaved by Cas12a to produce 2 guide RNAs for dual targeting of each protein coding gene, paralog targeting, and genetic interaction mapping. Bottom arrow: Upon intron splicing or intron retention, two different reads are generated. (B) Two different cell lines, stably expressing Cas9 and Cas12a, are used: HAP1, and RPE1. The pipeline before RNA-seq is depicted. (C) Heatmap showing single genes only (after removing gene pairs), whether identified individually or as part of a gene pair. The hits are ordered by the average ΔPIR values across HAP1 and RPE1, from lowest to highest. (D) Left: Gene ontology enrichment analysis of biological processes regulated by the hits of the screen. The most significant pathways involve mRNA processing and RNA splicing. Right: Gene ontology enrichment analysis of cellular components regulated by the hits of the screen. These components include mainly Spliceosomal Complex, small nuclear ribonucleoprotein complex and the Catalytic step 2 Spliceosome. (E) Venn diagram showing that there are 5 common hits which cause a reduction in the percentage of intron retention (ΔPIR) of the splicing reporter in the two cell lines. (F) Table showing the top 5 hits of the screen with a ΔPIR for each hit in each cell line and their full names.

Article Snippet: For HCT116 MALAT1 knockout, we used integration by non-homologous end joining, which was accomplished by introducing a simultaneous double-strand break in genomic DNA and in the targeting vectors (pCMV-Puro and pCMV-Hygro)., Plasmids encoding spCas9 and sgRNAs were obtained from Addgene (Plasmids #41815 and #47108).

Techniques: Plasmid Preparation, Selection, Genomic Sequencing, Construct, Negative Control, Generated, Stable Transfection, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Chemical control of 2’-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

doi: 10.64898/2026.01.26.701763

Figure Lengend Snippet: ( A ) Schematic of the crRNA sequence used for targeting an EGFP reporter gene and chemical modifications used to probe 2’-OH contacts. In vitro cleavage activity (orange) and cell-based EGFP editing (blue) is shown on the right. 2’-OH contacts with SpCas9 are indicated with red asterisks below. n = 3 or more experimental replicates. Error bars are S.E.M. ( B ) Time-course of in vitro cleavage activity using select crRNAs from panel A. Curves were fitted to an exponential two-phase decay equation. Error is reported as S.E.M. ( C ) Thermal denaturation of Cas9 RNP complexes assayed by absorbance at 280 nm to measure melting temperature ( T m ). n = 2 experimental replicates. Error bars are S.E.M. ( D ) Target DNA binding by dCas9 RNP measured by dot blot filter binding of radiolabeled target DNA. Curves were fit to a one-site binding curve. n = 2 experimental replicates. Error bars are S.E.M. ( E ) CRISPRa-based assay to measure dCas9-VPR binding guided by crTREa crRNA to a Tet-On 3G promoter driving EGFP in HeLa cells. Unmodified crTREa is shown as a control. EGFP expression was quantified by flow cytometry. N = 3 experimental replicates, Error is S.E.M.

Article Snippet: pET-Cas9-NLS-6xHis (Addgene #62933) plasmid encoding SpCas9, or a catalytically inactive “dead” Cas9 (dCas9) plasmidprepared by site-directed mutagenesis to generate H840A and D10A mutations (pET-dCas9-NLS-6xHis), were used.

Techniques: Sequencing, In Vitro, Activity Assay, Binding Assay, Dot Blot, Control, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Chemical control of 2’-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

doi: 10.64898/2026.01.26.701763

Figure Lengend Snippet: ( A , B ) Cryo EM reconstructions of SpCas9 ternary complexes assembled with ( A ) unmodified crRNA crE2 or ( B ) 2′ modified crEG-SJ1. Models are colored by atomic B factor from blue (low) to red (high). ( C ) Superposition of the two ternary complexes, with the unmodified (crE2) complex in magenta and the modified (crEG-SJ1) complex in green. Left, full ternary complexes; middle, Cas9 proteins only; right, crRNAs only. ( D ) Close up views of individual Cas9 domains from the superimposed structures, highlighting minimal local perturbations in RuvC, HNH, bridge helix (BH), PI, REC I, REC II and REC III. Unmodified and modified complexes are shown in magenta and green, respectively.

Article Snippet: pET-Cas9-NLS-6xHis (Addgene #62933) plasmid encoding SpCas9, or a catalytically inactive “dead” Cas9 (dCas9) plasmidprepared by site-directed mutagenesis to generate H840A and D10A mutations (pET-dCas9-NLS-6xHis), were used.

Techniques: Cryo-EM Sample Prep, Modification