Journal: Current biology : CB

Article Title: Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns

doi: 10.1016/j.cub.2019.12.067

Figure Lengend Snippet: (A) RNAi of RTO in M. lewisii generates a range of anthocyanin spot patterns. (B) RNAi of MgRTO in M. guttatus recapitulates the rto-like phenotype. (C) Over-expression of RTO in M. lewisii abolishes anthocyanin production throughout the corolla. (D–G) Relative expression of NEGAN and RTO in M. lewisii RTO over-expression lines (D), M. lewisii RTO RNAi lines (E), M. guttatus RTO RNAi lines (F), and M. guttatus CRISPR/Cas9 mediated knockout lines (G). All relative transcript levels are measured by qRT-PCR, standardized to the corresponding wild-type (LF10 for M. lewisii, MAC for M. guttatus). Error bars represent 1 SD from three biological replicates. (H) BiFC assay shows that the wild-type RTO protein interacts with ANbHLH1, whereas the D>G amino acid replacement in the mutant rto protein abolishes or attenuates the interaction. See also Figures S2 and S4–S6 and Tables S5 and S6.

Article Snippet: S. pyogenes Cas9 nuclease , New England Biolabs , Cat#M0386S.

Techniques: Over Expression, Expressing, CRISPR, Knock-Out, Quantitative RT-PCR, Bimolecular Fluorescence Complementation Assay, Mutagenesis

Journal: Current biology : CB

Article Title: Two MYB proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns

doi: 10.1016/j.cub.2019.12.067

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: S. pyogenes Cas9 nuclease , New England Biolabs , Cat#M0386S.

Techniques: Recombinant, Multiplex Assay, SYBR Green Assay, Sequencing, Generated, Clone Assay, Software

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: Glycan substrates and relative activity for Endo S and Endo S2.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: Activity Assay

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: In all cases, digestions were performed at 37 °C for 20 min with 0.5 μg of the indicated enzyme in 20 μL buffer of 50 mM MES pH 6.0 and separated on 17% SDS-gel. Both glycan fluorescent images and protein images of the same gels are shown. N2f was run in all three groups and served as a control. a General scheme for Endo S/S2 and Endo-β-Gal digestions. Digestion by Endo S/S2 results in the product Fuc-α,6-GlcNAc and digestion by Endo-β-Gal results in product N2f. b Digestion of Group I glycans by all three enzymes. While Endo S2 showed complete digestion on all substrates, Endo S only showed complete digestion on N2f and G2f. Endo-β-Gal only digested xG2f and xxG2f. c Digestion of Group II glycans by Endo S/S2. Slight product formation was observed when M1N1f was digested by Endo S and M2f was digested by Endo S2. d Digestion of Group III glycans by all three enzymes. No obvious digestion was observed on these substrates. Product formation in N3f digestion by Endo S/S2 was due to the digestion on contaminated N2f that was due to incomplete conversion during synthesis.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: SDS-Gel

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: In each reaction, 2 pmol of a substrate glycan was digested with 500 ng enzyme in 20 μl buffer of indicated pH for 30 min at 37 °C and then separated on 15% gel. Both glycan images and protein images are shown for the assays. a pH profile of Endo S on G2f. b pH profile of Endo S2 on G2f. c pH profile of Endo-β-Gal on xG2f.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques:

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: Different glycans were digested with indicated amounts of Endo S/S2 and separated on 15% gels and imaged for both glycans and proteins. The RA of each measurement are shown. a Relative activity on M1N1f. b Relative activity on N2f. c Relative activity on G2f. d Relative activity on N2nf. Except d where samples were digested for 18 h, all other samples were digested for 1 h at 37 °C.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: Activity Assay

Journal: Communications Biology

Article Title: Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities

doi: 10.1038/s42003-022-03444-3

Figure Lengend Snippet: a Plot of relative activities of Endo S/S2 on various glycans. Endo S/S2 are most active on N2f and G2f and are much less active on all other glycans. b Endo S/S2 substrate recognition motifs. The impacts of individual monosaccharide modification introduced by various glycosyltransferases on the activity of Endo S/S2 are indicated with arrows, x symbols and folds of increase/decrease. The key residues identified from this study are numbered and possible enzyme recognition motif is highlighted.

Article Snippet: Recombinant Streptococcus pyogenes endoglycosidase Endo S (Gene # AAK00850) and Endo S2 (Gene # number: ACI61688.1, Cat# 10976-GH), recombinant Flavobacterium keratolyticus Endo-β-Galactosidase (Endo-β-Gal) (Gene # Q9ZG90, Cat# 8620-GH), recombinant human FUT8 (Cat# 5768-GT), MGAT1 (Cat# 8334-GT), MGAT3 (Cat# 7359-GT), MGAT5 (Cat# 5469-GT), ST3Gal6 (Cat# 10591-GT), ST6Gal1 (Cat# 7620-GT), B3GNT2 (Cat# 3960-GT), B4GalT1 (Cat# 3609-GT) and HEXA (Cat# 6237-GH), and GDP-Cy5-Fuc (Cat# ES301) were from Bio-techne.

Techniques: Modification, Activity Assay

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac15 - Ac16 in Bac-eGFP/HRPc. ( A ) Schematic representation of the Ac15 - Ac16 genomic locus before and after CRISPR editing. The Ac15 and Ac16 are represented in green and purple, respectively. After deletion of the Ac15 -Ac 16 fragment, the resulting pseudogene consists of the 5′ portion of Ac15 (green) and the 3′ portion of Ac16 (purple). Gene-specific primers used for PCR are indicated by black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses (+/−), using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac15 and Rv- Ac16 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (2033 bp). The lane labeled “ Ac-eGFP / HRPc ΔAc15-Ac16 ” corresponds to the PCR product amplified from the edited virus (749 bp). Marker: Trans 2K Plus (TransGen Biotech, Beijin, China). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. ( D ) Amino acid sequence of the truncated aberrant protein originating from the fusion of Ac15 and Ac16 . The conserved N-terminal region from Ac15 is shown in green, followed by a frameshift-derived sequence (i.e., a sequence resulting from a mutation that alters the original reading frame) leading to a premature stop codon (*). Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Marker, Sequencing, Derivative Assay, Mutagenesis

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac129 - Ac131 in Bac-eGFP/HRPc. ( A ) Schematic representation of Ac129 - Ac131 genomic locus before and after CRISPR editing. The non-edited Ac128 is shown as a dark grey arrow, while Ac129 , Ac130 , and Ac131 are represented in green, light gray, and purple, respectively. After deletion of the Ac129-Ac131 fragment, the resulting pseudogene consists of the 5′ portion of Ac129 (green) and the 3′ portion of Ac131 (purple). Ac130 (light grey) is completely removed. Gene-specific primers used for PCR are indicated by black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses (+/−), using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac129 and Rv- Ac131 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (1733 bp). The lane labeled “ Ac-eGFP/HRPc ΔAc129-Ac131 ” corresponds to the PCR product amplified from the edited virus (569 bp). Marker: Trans 2K Plus (TransGen Biotech). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. A single nucleotide insertion (T) at the fusion is indicated with a square. ( D ) Amino acid sequence of the truncated aberrant protein originating from the junction of Ac129 and Ac131 . The conserved N-terminal region from Ac129 is shown in green, followed by a frameshift-derived sequence that leads to a premature stop codon (*). Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Marker, Sequencing, Derivative Assay

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac136 - Ac138 in Bac-eGFP/HRPc. ( A ) Schematic representation of the Ac136-Ac138 genomic locus before and after CRISPR editing. The non-edited HR5 is shown as a dark grey arrow, while Ac136 , Ac137 , and Ac138 are represented in green, light gray, and purple, respectively. After deletion of the Ac136 -Ac 138 fragment, the resulting pseudogene consists of the 5′ portion of Ac136 (green) and the 3′ portion of Ac138 (purple). Ac137 (light grey) is completely removed. Gene-specific primers used for PCR are shown as black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses (+/−), using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac136 and Rv- Ac138 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (3134 bp). The lane labeled “ Ac-eGFP/HRPc ΔAc129-Ac131 ” corresponds to the PCR product amplified from the edited virus (379 bp). Marker: Trans 2K Plus (TransGen Biotech). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. A single nucleotide insertion (c) at the junction is indicated with a square. ( D ) Amino acid sequence of the truncated aberrant protein originating from the fusion of Ac13 6 and Ac138 . 1. The conserved N-terminal region from Ac129 is shown in green, followed by a frameshift-derived sequence that leads to a premature stop codon (*). 2. The conserved N-terminal region from Ac138 is shown in green, followed by a frameshift-derived sequence that leads to a premature stop codon (*). Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Marker, Sequencing, Derivative Assay

Journal: Viruses

Article Title: CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production

doi: 10.3390/v17081041

Figure Lengend Snippet: CRISPR dual-sgRNA knockout of Ac148 -Ac 150 in Bac-eGFP/HRPc. ( A ) Schematic representation of Ac148 -Ac 150 genomic locus before and after CRISPR editing. The edited Ac148 , Ac149 , and Ac150 are represented in green, light gray and purple, respectively. After deletion of the Ac148-Ac150 fragment, the resulting pseudogene is composed of the 5′ portion of Ac148 (green) and the 3′ portion of Ac150 (purple). Ac149 (light grey) is completely removed. Gene-specific primers used for PCR are indicated by black arrows. DNA excision is marked with scissors and a triangle (▲). The hybridization positions of primers and sgRNA targets are shown in parentheses, using the ATG start codon of each ORF as the reference point. PAM sequences (red nucleotides) are highlighted. ( B ) Identification of edited clones by PCR using specific primers (Fw- Ac148 and Rv- Ac150 ). The lane labeled “ Ac-eGFP/HRPc ” corresponds to the PCR product amplified from the unedited parental virus (886 bp). The lane labeled “ Ac-eGFP/HRPc ΔAc12-Ac131 ” corresponds to the PCR product amplified from the edited mutant virus (367 bp). Marker: Trans 2K Plus (TransGen Biotech). ( C ) Sanger sequencing chromatogram of the edited virus. Only the flanking regions of the knockout are shown. Not at scale.

Article Snippet: All were synthesized using the Engen ® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s protocols.

Techniques: CRISPR, Knock-Out, Hybridization, Clone Assay, Labeling, Amplification, Virus, Mutagenesis, Marker, Sequencing

Journal: iScience

Article Title: Programmable modulation of ribosomal frameshifting by mRNA targeting CRISPR-Cas12a system

doi: 10.1016/j.isci.2023.108492

Figure Lengend Snippet:

Article Snippet: E. coli Acella cells containing the MBP-dCas9 (Addgene, #60815 ) were grown in the 2 L of LB medium supplemented with 0.5% glucose and 50 μg/mL Ampicillin at 18°C.

Techniques: Virus, Recombinant, Cloning, Luciferase, Software

Journal: Neoplasia (New York, N.Y.)

Article Title: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma

doi: 10.1016/j.neo.2025.101139

Figure Lengend Snippet: Generation of primary DMGs in mice using CRISPR/Cas9. (A) Post-natal day 3-5 NNC mice were intracranially injected with virus-secreting DF1 chicken fibroblast cells transfected with RCAS-Trp53-gRNA-BFP and RCAS-Cntl-gRNA-PDGFB plasmids. (B) Kaplan-Meier survival analysis of mice that received and did not receive intracranial injections of virus-secreting fibroblast cells on post-natal day 3-5. P-value for log-rank test comparison between groups. (C) Whole mount (top) and H&E slides showing tumor (bottom left) and non-tumor (bottom right) of NNC mice. Scale bar for whole mount H& E = 2 mm. Scale bar for H& E = 50 µm; images taken at 40x. (D) IHC images of mouse DMGs in NNC mice incubated with anti-Ki67, anti-HA, anti-Cas9, anti-GFP, or anti-p53 antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x. (E) IHC images of non-tumor brain tissue in NNC mice incubated with anti-Ki67, anti-HA, anti-Cas9, anti-GFP, or anti-p53 antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x.

Article Snippet: Antibodies used were rabbit monoclonal Cas9 primary antibody (#ab189380), rabbit monoclonal GFP primary antibody (#ab183734), rabbit monoclonal HA-Tag primary antibody (Cell Signaling #3724), rabbit polyclonal Ki67 primary antibody (#ab15580), rabbit polyclonal p53 primary antibody (#NCL-l-P53CM5P), and rabbit polyclonal PTEN primary antibody (#AF847).

Techniques: CRISPR, Injection, Virus, Transfection, Comparison, Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma

doi: 10.1016/j.neo.2025.101139

Figure Lengend Snippet: Two approaches to generate tumors containing CRISPR/Cas9 perturbations. (A) On the left, Cre-recombinase is expressed from an endogenous Nestin-Cre allele in the germline to drive Cas9 expression in all Nestin+ neural stem cells (NNC mice), allowing the use of only 2 viral constructs. On the right, Cre is delivered via a third retroviral construct (NC mice). (B) Anti-Cas9 IHC for NNC mice brain tissue. Scale bar for whole mount IHC = 2 mm. Scale bar for IHC of tumor and non-tumor tissue = 50 µm; images taken at 40x. (C) Anti-Cas9 IHC for NC mice brain tissue. Scale bar for whole mount IHC = 2 mm. Scale bar for IHC of tumor and non-tumor tissue = 50 µm; images taken at 40x. (D) Tumor penetrance and median time to tumor formation for NNC and NC mice.

Article Snippet: Antibodies used were rabbit monoclonal Cas9 primary antibody (#ab189380), rabbit monoclonal GFP primary antibody (#ab183734), rabbit monoclonal HA-Tag primary antibody (Cell Signaling #3724), rabbit polyclonal Ki67 primary antibody (#ab15580), rabbit polyclonal p53 primary antibody (#NCL-l-P53CM5P), and rabbit polyclonal PTEN primary antibody (#AF847).

Techniques: CRISPR, Expressing, Construct, Retroviral

Journal: Neoplasia (New York, N.Y.)

Article Title: Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma

doi: 10.1016/j.neo.2025.101139

Figure Lengend Snippet: Pooled in vivo CRISPR experiment. (A) Post-natal day 3-5 NNC mice were intracranially injected with virus-secreting DF1 chicken fibroblast cells transfected with RCAS-gRNA-PDGFB plasmids targeting Atm, Cdkn2a, Pten, and Trp53 respectively. (B) Kaplan-Meier analysis of mice monitored for survival following injection (mice analyzed in are shown for reference, P-value for log-rank test shown). (C) Whole mount (top) and H&E slides showing tumor (bottom left) and non-tumor (bottom right) of NNC mice. Scale bar for whole mount H& E = 2 mm. Scale bar for H& E = 50 µm; images taken at 40x. (D) IHC images of NNC mouse midline gliomas incubated with anti-Ki67, anti-Cas9, anti-p53, and anti-Pten antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x. (E) IHC images of NNC mouse non-tumor brain tissue incubated with anti-Ki67, anti-Cas9, anti-p53, and anti-Pten antibody and biotinylated secondary antibody. Scale bar for IHC images = 50 µm; images taken at 40x.

Article Snippet: Antibodies used were rabbit monoclonal Cas9 primary antibody (#ab189380), rabbit monoclonal GFP primary antibody (#ab183734), rabbit monoclonal HA-Tag primary antibody (Cell Signaling #3724), rabbit polyclonal Ki67 primary antibody (#ab15580), rabbit polyclonal p53 primary antibody (#NCL-l-P53CM5P), and rabbit polyclonal PTEN primary antibody (#AF847).

Techniques: In Vivo, CRISPR, Injection, Virus, Transfection, Incubation

Journal: bioRxiv

Article Title: Prime editing of the β 1 adrenoceptor in the brain reprograms mouse behavior

doi: 10.1101/2023.05.19.541410

Figure Lengend Snippet: ( a ) Schematic representation of AAV designs used in vivo and their corresponding lengths in kilobase pairs (kbp, including ITRs) for neuron-specific expression of PEmax or PE3bmax. Constructs are not depicted to scale. ( b ) Schematic representation of the experimental setup and timeline. ( c ) In vivo prime editing and indel rates of different AAV vector designs in mouse cortices at 5 weeks, 10 weeks and 6 months post-injection. ( d ) Editing and indel rates at the Adrb1 (AAV-PE3bmax-nT and W3-synth-cT) and Dnmt1 (AAV-PEmax-nT and noW3-bGH-cT) locus in different brain regions at 10 weeks post-injection. ( e ) Frequency of Adrb1 and Dnmt1 edits in various other tissues in saline-and phsyn-PE-treated animals at 6 months post injection. Animals were treated with the same AAV preparations as in (d). Skeletal muscle tissue was isolated from the quadriceps femoris. ( f ) Editing rates at the Dnmt1 locus in mouse cortices (5 and 10 weeks post-injection), liver, and heart (10 weeks post-injection) after ICV injection. Animals were treated with PEmax-noW3-synth-nT and noW3-synth-cT under the control of the ubiquitous Cbh promoter . ( g ) Comparison of editing and indel rates at the Adrb1 locus for PEmax complexed with epegRNA1 or epegRNA1-SM CTT (with and without a PE3b-ngRNA) at 5 weeks. Animals were treated with AAV-PEmax-nT and AAV-W3-synth-cT. Data are displayed as means±s.d. of at least three animals. Each data point represents one animal. ITR, inverted terminal repeat; nT/cT, N-/C-terminal PEmax AAV vector; phsyn, human synapsin promoter; NLS, nuclear localization signal; n Sp Cas9, Sp Cas9 nickase; M-MLV, Moloney Murine Leukemia virus; W3, woodchuck hepatitis virus post-transcriptional regulatory element; hU6/mU6, human/mouse U6 promoter; SV40, Simian virus 40; pA, polyA signal; synth, synthetic polyA signal; bGH, bovine growth hormone polyA signal; kbp, kilobasepairs; vg, vector genomes; wks, weeks; m, months; SM, silent mutation; pCbh, truncated chimeric CMV/chicken-β-actin hybrid promoter.

Article Snippet: Sections were blocked in PBS supplemented with 2% normal donkey serum (cat. no. ab7475, abcam) and 0.3% Triton X-100 (Sigma-Aldrich) for 1 h. Brain sections were incubated with primary antibodies overnight at 4°C (mouse-NeuN, 1:500, abcam ab177487; rabbit-Cas9, 1:1,000, Cell Signaling clone D8Y4K; chicken-GFAP, 1:1’500, abcam ab95231).

Techniques: In Vivo, Expressing, Construct, Plasmid Preparation, Injection, Saline, Isolation, Control, Comparison, Virus, Mutagenesis