Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Production and Characterization of Cas9 Gesicles

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Assaying Gesicle Size and Concentration

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques: Concentration Assay

Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Gesicle Treatment Rapidly Delivers Protein to CHME-5 Microglia

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Schematic of the HIV Long Terminal Repeat and Expected Products after CRISPR/Cas9 Targeting by Gesicles

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques: CRISPR

Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Gesicle Treatment Causes Specific Mutation Events at the HIV LTR

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques: Mutagenesis

Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Gesicle Treatment Reduces HIV Proviral Activity

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques: Activity Assay

Journal: Molecular Therapy

Article Title: Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus

doi: 10.1016/j.ymthe.2018.10.002

Figure Lengend Snippet: Gesicle Dosage and Viability

Article Snippet: Unless otherwise noted, gesicles were prepared using the Guide-it CRISPR/Cas9 Gesicle Production System (catalog [Cat.] no. 631613; Clontech/Takara, Mountain View, CA, USA) according to the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: Microglia ferroptosis is prevalent in neurodegenerative disease and regulated by SEC24B

doi: 10.1101/2021.11.02.466996

Figure Lengend Snippet: (A) Schema for positive selection Genome-wide CRISPR screen in Cas9-expressing human immortalized microglia. (B) Venn diagrams for hits from each viral pool in 8hr and 24hr treatments. (C) Overlapping hits from each viral pool with number of gRNAs identified in 8hr and 24hr treatment with SEC24B and ACSL4 highlighted. (D) qRT-PCR analysis showing markedly reduce expression of SEC24B in KO line (n=3). Unpaired t test. *p<0.05. Error bars represent SEM. (E) Western blot showing absence of SEC24B protein in KO line. (F) Death kinetics in SEC24B KO Hap1 cell line and isogenic control (n=3). AUC, one-way ANOVA, Dunnett post hoc. ****p<0.0001. Error bars represent SEM.

Article Snippet: The sgRNA sequences were amplified using Guide-it™ CRISPR Genome-Wide Library PCR Kit (Takara, 632651) and subjected to the high-throughput amplicon sequencing on NextSeq500.

Techniques: Selection, Genome Wide, CRISPR, Expressing, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: An engineered biosensor enables dynamic aspartate measurements in living cells

doi: 10.7554/eLife.90024

Figure Lengend Snippet: CRISPR guides.

Article Snippet: Sequence-based reagent , GOT1_1 , Synthego , CRISPR guide , CAGUCAUCCGUGCGAUAUGC.

Techniques: CRISPR, Sequencing

Journal: eLife

Article Title: An engineered biosensor enables dynamic aspartate measurements in living cells

doi: 10.7554/eLife.90024

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , GOT1_1 , Synthego , CRISPR guide , CAGUCAUCCGUGCGAUAUGC.

Techniques: Virus, Sequencing, CRISPR

Journal: Genes & Development

Article Title: A C9orf72–CARM1 axis regulates lipid metabolism under glucose starvation-induced nutrient stress

doi: 10.1101/gad.315564.118

Figure Lengend Snippet: Loss of C9orf72 increases FA flux and biogenesis. ( A , B ) Wild-type and C9KO MEFs were starved with glucose-free medium for 16 h, and then the association of autophagy with LDs was monitored by immunostaining of BODIPY and lysosomal marker LAMP1. The number of BODIPY and LAMP1 double-positive puncta per cell was counted, and 30–40 cells in each group from three independent experiments were statistically analyzed. ( C ) Wild-type and C9KO MEFs were grown with or without glucose starvation for 16 h, and then LDs were isolated and subjected to SDS-PAGE and immunoblot against anti-LC3 and P62. ADRP served as a loading control. The ratios of LC3II/I and the amounts of P62 were calculated and statistically analyzed. n = 3. ( D – F ) Wild-type, C9KO, and C9/Atg5 double-knockout MEFs were starved with glucose-free medium for 16 h. The LDs were labeled with BODIPY and counted, and the association of autophagy and LDs was monitored as in A . Twenty-eight to 30 cells in each group from three independent experiments were statistically analyzed. ( G ) Wild-type and C9KO MEFs were cultured in CM or glucose-free medium with [1,2- 14 C] acetate at a final concentration of 0.3 µCi for 6 h. The incorporation of [1,2- 14 C] in total lipids was measured by a scintillation counter. Counts per minute (CPM) were normalized to the total amount of protein. n = 6. ( H – J ) Wild-type and C9KO MEFs were grown in glucose-free medium, and the protein levels of acetyl-CoA carboxylase (ACC) and NOX2 were determined at the indicated time points by immunoblotting. n = 3. (GS) Glucose starvation. Data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01.

Article Snippet: The guide RNAs (gRNAs) for CRISPR–Cas9-mediated knockout of CARM1 or Atg5 were designed with Benchling as follows: CARM1 (oligo1: 5′-CACCGCTCACTATCGGCGACGCGAA-3′; oligo2: 5′-AAACTTCGCGTCGCCGATAGTGAGC-3′) and atg5 (oligo1: 5′-CACCGAAGAGTCAGCTATTTGACGT-3′; oligo2: 5′-AAACACGTCAAATAGCTGACTCTTC-3′).

Techniques: Immunostaining, Marker, Isolation, SDS Page, Western Blot, Control, Double Knockout, Labeling, Cell Culture, Concentration Assay