Journal: Lipids in Health and Disease

Article Title: Sphingomyelin synthase overexpression increases cholesterol accumulation and decreases cholesterol secretion in liver cells

doi: 10.1186/1476-511X-10-46

Figure Lengend Snippet: The expression of ABCA1, ABCG1 and SR-BI . A . RT-PCR analysis; B . Western blot analysis. Values shown are means ± SD ( n = 3), * P < 0.05; ** P < 0.001 compared to control (Huh7-tTA).

Article Snippet: Proteins were electroblotted to a nitrocellulose membrane that had been incubated with respective antibodies: FLAG (CST), Apo A-I (Epitomics), ABCA1 (Boster), ABCG1 (Boster), or SR-BI (Epitomics).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: bioRxiv

Article Title: Screening in serum-derived medium reveals differential response to compounds targeting metabolism

doi: 10.1101/2023.02.25.529972

Figure Lengend Snippet:

Article Snippet: 100% adult bovine serum (Sigma-Aldrich B9433, lot 16A041) containing pen/strep was pumped through a Vivaflow 10,000 MWCO Hydrosart membrane (Sartorius VF20H0) according to manufacturer recommendations using a Masterflex L/S Easy-Load II peristaltic pump (Masterflex HV-77200-50) at 4°C.

Techniques: Recombinant, Labeling, Generated, Cell Viability Assay, Software, Membrane

Journal: Cell reports

Article Title: Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs

doi: 10.1016/j.celrep.2024.113777

Figure Lengend Snippet:

Article Snippet: Plasmids for mammalian cell expression of Cas12a were gifted from various labs (Zhang, Kleinstiver, Li, and Ekker)., , , , The plasmid encoding crRNAs under U6 promoter was obtained from Joung and Kleinstiver labs (Addgene #114087) and subsequently modified at the scaffold region to create the nine distinct crRNAs via site-directed mutagenesis.

Techniques: Virus, Recombinant, Protease Inhibitor, DNA Extraction, Mutagenesis, Nucleic Acid Purification, Cloning, Plasmid Preparation, Software

Journal: Genome Research

Article Title: Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

doi: 10.1101/gr.255414.119

Figure Lengend Snippet: Functional PAM sequences for robust and potent DNA cleavage by St1Cas9 LMD9 in mammalian cells. ( A ) Schematic representations of St1Cas9 LMD9 flanked by nuclear localization signals (NLSs) and its engineered sgRNA (v1). Nucleotide sequence and functional modules are depicted; crRNA (green), loop (gray), tracrRNA (blue), and mutated nucleotides (orange). ( B ) K562 cells stably expressing St1Cas9 were transfected with indicated sgRNA expression vectors at increasing doses, and TIDE assays were performed 3 d later to determine the frequency of indels. An expression vector encoding EGFP (−) was used as a negative control. The experiment was performed twice and yielded equivalent results; only one is shown. ( C ) Screening for guides targeting St1Cas9 LMD9 to various PAMs was performed by transient transfections in K562 (solid shapes) and Neuro-2a (open shapes) cells using single-vector constructs driving the expression of St1Cas9 and its sgRNA. Surveyor assays were performed 3 d later to determine the frequency of indels. An expression vector encoding EGFP (−) was used as a negative control. See also Supplemental Figure S1 .

Article Snippet: BPK2301 (v0; Addgene plasmid 65778, a gift from Keith Joung) ( ) was used to compare St1Cas9 sgRNA architectures.

Techniques: Functional Assay, Sequencing, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Negative Control, Construct

Journal: Genome Research

Article Title: Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

doi: 10.1101/gr.255414.119

Figure Lengend Snippet: Structural basis for PAM specificity of engineered St1Cas9 variants with expanded targeting range. ( A ) Schematic representation of St1Cas9 hybrid proteins containing the N terminus of LMD9 and the C-terminal domains (WED + PI) of LMG18311 or CNRZ1066. To determine the activity of St1Cas9 variants programmed with sgRNAs compatible with different PAMs, K562 cells were transiently transfected with single-vector constructs driving expression of St1Cas9 and its sgRNA. For each PAM and nuclease combination, four different sgRNAs (targets) were tested. Surveyor assays were performed 3 d later to determine the frequency of indels. An expression vector encoding EGFP (−) was used as a negative control. The experiment was performed twice and yielded equivalent results; only one is shown. ( B ) Close-up view of the 5′-GCAGAAA-3′ PAM bound to the St1Cas9 (DGCC7010) PI domain (PDB: 6RJD). The target (turquoise) and nontarget (blue) strands are shown as sticks (the phosphate–sugar backbones are also shown as ribbons). The ribbon representation of the PI domain is orange. The hydrogen bonds between the side chain of St1Cas9 K1086 and the nucleobase of dG4 is shown as a dashed line. ( C , D ) The PI domains of St1Cas9 and SaCas9 (PDB: 5CZZ, gray ribbon) are superimposed. ( C ) The St1Cas9 Q1084 and SaCas9 R1015 occupy the same positions relative to the PAM (dA3). The St1Cas9 E1057 and SaCas9 E993 occupy the same positions relative to St1Cas9 Q1084 and SaCas9 R1015, respectively. ( D ) St1Cas9 T1048 and M1049 (substituted for N1048 and D1049 in some variants) superimpose onto SaCas9 N985 and N986 that specify purines in positions 4 and 5 of the PAM. See also Supplemental Figure S3 .

Article Snippet: BPK2301 (v0; Addgene plasmid 65778, a gift from Keith Joung) ( ) was used to compare St1Cas9 sgRNA architectures.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Construct, Expressing, Negative Control

Journal: Genome Research

Article Title: Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

doi: 10.1101/gr.255414.119

Figure Lengend Snippet: Broadening the targeting scope of base editors using St1Cas9 variants. ( A ) K562 cells were transiently transfected with single-vector constructs driving expression of St1BE4max LMD9 and its sgRNA. Genomic DNA was harvested 3 d later, and quantification of base editing was performed on PCR amplified target sites using EditR. The target sequence was defined as the 20 bases upstream of the PAM and are numbered in decreasing order from the PAM. Sequence of the guides and related PAMs are shown with target cytosine highlighted in blue. An expression vector encoding EGFP (−) was used as a negative control. ( B – D ) Same as A but using St1BE4max LMG18311, CNRZ1066, and TH1477 chimeric proteins. ( E ) Same as A but using St1ABEmax LMD9. Target adenines are highlighted in red. Most sgRNAs were tested at least twice; only one experiment is shown. See also Supplemental Figure S5 .

Article Snippet: BPK2301 (v0; Addgene plasmid 65778, a gift from Keith Joung) ( ) was used to compare St1Cas9 sgRNA architectures.

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Amplification, Sequencing, Negative Control

Journal: Genome Research

Article Title: Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

doi: 10.1101/gr.255414.119

Figure Lengend Snippet: In vivo genome editing using St1Cas9. ( A ) The tyrosine degradation pathway and associated inborn errors of metabolism (IEMs). ( B ) Experimental design. Neonatal (2-d-old) Fah −/− mice were injected with AAV8-St1Cas9 or saline into the retro-orbital sinus and weaned at 21 d, and NTBC was removed at 30 d of age. Mice off NTBC were sacrificed when they lost 20% of their body weight. ( C ) Schematic representation of the AAV-St1Cas9 v3 vector. Annotated are the liver-specific promoter (LP1b), synthetic polyadenylation sequence (SpA), and hU6 promoter. Arrows indicate the direction of transcriptional unit. ( D ) Neonatal Fah −/− mice were injected with either 5 × 10 10 or 1 × 10 11 vector genomes (vg) of AAV8-St1Cas9 v3 targeting Hpd exon 13 and sacrificed 28 d following injection or kept alive for phenotypic and metabolic studies for 4 mo post NTBC removal. Genomic DNA was extracted from whole-liver samples, and the Surveyor assay was used to determine the frequency of indels. Each dot represents a different mouse. A mouse injected with saline (−) was used as a negative control. ( E ) SUAC levels in urine from treated mice were determined 15 d (short term) or 4 mo (long term) following NTBC removal. Samples were collected from the indicated treatment groups over a 24-h period using metabolic cages. Number of mice per group/metabolic cage (n) and AAV doses (vg) is indicated. SUAC levels are undetectable in C57BL/6N (wild-type) mice. ( F – H ) Survival analysis, body weight, and glycemia following NTBC removal in treated mice. Body weight was measured daily, and glycemia was monitored in nonfasted mice. Solid lines designate the mean; and error bars are represented by shaded areas and denote SEM. See also Supplemental Figure S6 .

Article Snippet: BPK2301 (v0; Addgene plasmid 65778, a gift from Keith Joung) ( ) was used to compare St1Cas9 sgRNA architectures.

Techniques: In Vivo, Injection, Saline, Plasmid Preparation, Sequencing, Negative Control