Journal: Molecular Therapy

Article Title: In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation

doi: 10.1016/j.ymthe.2018.04.017

Figure Lengend Snippet: Versatile Genome Regulation via a Modular Dual-AAV Split-dCas9 System

Article Snippet: Split-Cas9/dCas9 AAV vectors were constructed by sequential assembly of corresponding gene blocks (Integrated DNA Technologies) into a custom synthesized rAAV2 vector backbone. gRNA sequences were inserted into NCas9 or dNCas9 plasmids by cloning oligonucleotides (IDT) encoding spacers into AgeI cloning sites via Gibson assembly.

Techniques:

Journal: Molecular Therapy

Article Title: In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation

doi: 10.1016/j.ymthe.2018.04.017

Figure Lengend Snippet: Dual-AAV Split-dCas9 Repression Strategy Rescues Retinal Function in rd10 Mice

Article Snippet: Split-Cas9/dCas9 AAV vectors were constructed by sequential assembly of corresponding gene blocks (Integrated DNA Technologies) into a custom synthesized rAAV2 vector backbone. gRNA sequences were inserted into NCas9 or dNCas9 plasmids by cloning oligonucleotides (IDT) encoding spacers into AgeI cloning sites via Gibson assembly.

Techniques:

Journal: Bio-protocol

Article Title: Single-Particle Tracking of AMPA Receptor-Containing Vesicles

doi: 10.21769/BioProtoc.5325

Figure Lengend Snippet: (A) Constructs used to insert HaloTag into the genomic loci of Gria1 using homology-independent targeted integration (HITI). The donor construct (Donor) contains the DNA sequence coding for HaloTag (HaloTag) and the single guide RNA that will target Cas9 to Gria1 (sgRNA). Importantly, the HaloTag coding sequence is flanked by Gria1 sequences that will be targeted and cut by Cas9 (red bars). Cas9 is expressed from a second plasmid (Cas9). Black bars represent promoters driving the expression of Cas9 and the sgRNA. (B) Cas9 creates double-strand breaks in the genomic loci of Gria1 and releases HaloTag from the Donor. HaloTag can be inserted into Gria1 by non-homologous end joining (NHEJ). When expressed from the edited copy of Gria1 (Gria1-HT ), GluA1 will contain HaloTag inserted into its amino-terminal domain (ATD; GluA1-HT). SP, signal peptide; LBD, ligand-binding domain; TMD, transmembrane domain; CT, C-terminal tail. (C) Diagram of AMPAR containing a GluA1 subunit (blue) with HaloTag (green) inserted into the ATD (red). AMPARs are tetramers that can contain zero to four GluA1 subunits. As HaloTag is inserted into the ATD, it will sit on the extracellular side of the receptor. HaloTag can be labeled with fluorescent dyes that are conjugated to HaloTag ligand (HTL), such as JF 549 -HTL. (D) Representative confocal image of a cultured rat hippocampal neuron expressing GluA1 tagged with HaloTag and labeled with JF 549 -HTL (GluA1-HT-JF 549 ). Scale bar, 25 μm. (E) Representative widefield images of edited neurons expressing GluA1-HT labeled with JF 549 -HTL (GluA1-HT-JF 549 ) and a fluorescent neuron marker (in this case, miRFP670 driven by a synapsin promoter). Scale bar, 100 μm.

Article Snippet: PX551 (Cas9) plasmid (Addgene, catalog number: 60957) 3. px552-sg-gria1-HT (donor) plasmid (Addgene, catalog number: 187652) 4. rh10-PX551 AAV (Janelia Research Campus Viral Services) 5. rAAV2-px552-sg-gria1-HT AAV (Janelia Research Campus Viral Services) Reagents 1.

Techniques: Construct, Sequencing, Plasmid Preparation, Expressing, Non-Homologous End Joining, Ligand Binding Assay, Labeling, Cell Culture, Marker