Journal: Science translational medicine

Article Title: Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors

doi: 10.1126/scitranslmed.abh1486

Figure Lengend Snippet: (A and B) CRISPR-Cas9 FBXO42 knockout (sgFBXO42-3 and sgFBXO42-D2) and sgRNA control (sgControl) HCT116 clones were treated with HB007 for 72 hours and analyzed by Western blot for conjugated SUMO1 (A), dot blots for SUMO1 total amounts [(B) top], and cell viability assay for cell growth inhibition [(B) bottom)] (means ± SD; n = 3; ***P < 0.001 by unpaired t test). (C) Flag-FBXO42 and YFP-SUMO1-GV were cotransfected in HCT116 or LN229 cells. After 24 hours of treatment with HB007, the cells were subjected to Flag IP and Western blots using a GFP/YFP antibody for the interaction of FBXO42 and SUMO1. (D) Flag-CUL1, CUL2, or CUL3 was cotransfected with YFP-SUMO1-GV in HCT116 cells, and after treatment with HB007 for 48 hours, the cells were subjected to Flag IP and Western blots using GFP/YFP antibodies for the interaction of SUMO1 and CUL1, CUL2, or CUL3. (E) The FBXO42 knockout sg-FBXO42-3, sg-FBXO42-D2, and sgRNA control HCT116 clone were transfected with YFP-SUMO-GV and Flag-CUL1; treated with HB007 for 24 hours; and subjected to Flag IP and Western blot. (F) The sgRNA control and sg-FBXO42-D2 (left) or sg-FBXO42-3 HCT116 clone (right) was transfected with Flag-SUMO1-GV and HA-UB, treated with HB007 for 24 hours, and subjected to Flag IP and Western blot for SUMO1 polyubiquitination (top) and densitometry analysis of the HA-UB blots for the poly-UB amounts (bottom) (n = 2). (G) HCT116 cells were cotransfected with Flag-SUMO1-GV, Myc-FBXO42, and/or HA-UB; treated or untreated with HB007 for 24 hours; and subjected to Flag IP and Western blotting for SUMO1 polyubiquitination. (H) LN229 cells were treated with MLN4924 for 24 hours, alone or in combination with HB007, and analyzed by Western blots for conjugated SUMO1 and neddylated or unneddylated CUL1 as indicated (right).

Article Snippet: An all-in-one lentivirus plasmid vector was built comprising a selection marker (puromycin resistance), the expression cassette for Cas9, and sgRNA sequence and cloned by Gibson Assembly [New England BioLabs, #E2611S/L] in accordance with the manufacturer’s instructions.

Techniques: CRISPR, Knock-Out, Clone Assay, Western Blot, Viability Assay, Inhibition, Transfection

Journal: Science translational medicine

Article Title: Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors

doi: 10.1126/scitranslmed.abh1486

Figure Lengend Snippet: (A) Venn diagram of the data from genome-scale CRISPR-Cas9 knockout screen, HB007-FG bead and HB007-biotin/streptavidin-coated bead pull-down (top), and spectrometric total peptides counts of CAPRIN1 from LC-MS/MS analysis (bottom). (B) HB007-biotin was incubated with rhCAPRIN1 in the presence or absence of free HB007 and pulled down by streptavidin-coated beads and tested by immunoblotting for the binding of rhCAPRIN1 to HB007-biotin, with rhCAPRIN1 (5%) used as the loading control. (C) rhCAPRIN1 was premixed with 1 μM biotin, followed by HB007-biotin/streptavidin-coated bead pull-down in the presence of various doses of HB007. CAPRIN1 binding was identified by immunoblotting using CAPRIN1 antibodies. (D and E) HB007-FG beads and HB007-biotin were incubated with HCT116 lysate (D) and LN229 lysate (E) added or not with free HB007, and the pull-downs were tested by immunoblotting for the binding of cellular CAPRIN1 to HB007. (F) Immunoblot of HB007-biotin/streptavidin pull-down of HCT116 lysate added or not with the indicated concentrations of CPD1 or HB007. (G) The CPD1 similar but inactive compounds CID: 11208948 or CID: 789482 (top) or HB007 was added to HCT116 lysate that was then incubated with HB007-biotin. HB007-biotin/streptavidin pull-down was analyzed by immunoblotting. (H) Representative BLI sensorgrams of the interactions between rhCAPRIN1 and biotinylated HB007. Plots of the binding response during the association (0 to 600 s) and dissociation (600 to 1200 s) periods of the BLI assay at varying concentrations of CAPRIN1 (top) when HB007-biotin–loaded biosensors (quenched with biocytin) were dipped in CAPRIN1 wells. The plots have been processed with the double referencing technique and aligning of x and y axes. The red curves indicate the fit data. Residual binding for each plot at the varying concentrations (bottom). Binding curves were fit globally to a 1:1 binding model to calculate the binding constant (KD) from kinetic analysis as the ratio of the association (koff) and dissociation (kon) rate constants.

Article Snippet: An all-in-one lentivirus plasmid vector was built comprising a selection marker (puromycin resistance), the expression cassette for Cas9, and sgRNA sequence and cloned by Gibson Assembly [New England BioLabs, #E2611S/L] in accordance with the manufacturer’s instructions.

Techniques: CRISPR, Knock-Out, Liquid Chromatography with Mass Spectroscopy, Incubation, Western Blot, Binding Assay

Journal: Biotechnology and bioengineering

Article Title: Domesticating a food spoilage yeast into an organic acid-tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii.

doi: 10.1002/bit.27576

Figure Lengend Snippet: FIGURE 3 Zygosaccharomyces bailii ZbADE2 disruption experiment overview. Transfer RNA based maturation of gRNA (a). Design of ZbADE2 gRNA and donor DNA (b). Screening of ZbADE2 mutants (c), the mutants show distinctive red color accumulation in adenine‐deficient plate. Sequencing of ZbADE2 (d), the results show CRISPR Cas9 mediated mutation in ZbADE2 gene. gRNA, guide RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The original plasmid pUDP004 containing gRNA and Cas9 expression cassettes were obtained from ADDGENE (catalog #101165).

Techniques: Disruption, Sequencing, CRISPR, Mutagenesis

Journal: Science signaling

Article Title: A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses

doi: 10.1126/scisignal.aba3244

Figure Lengend Snippet: (A-C) qPCR (A), immunoblot (B) and flow cytometry plots (C) showing oncogene expression in THP-1 NOD1 cells in absence and presence of DOX. (D) Immunoblot showing the abundance of the oncogene C-MYC and the phosphorylation (p) of AKT in THP-1 NOD1 cells in presence or absence of DOX. (E) Kinetics of C-JUN and C-MYC expression following ligand treatment as measured by qPCR. Grey arrows indicate times of recurring ligand addition (0, 24 and 48 h). Expression at each time point is represented relative to the untreated control at that timepoint (dashed line set to 1). (F-H) qPCR (F) and immunoblots (G, H) for the indicated genes in THP-1 NOD1 cells following CRISPR/Cas9 mediated ablation of FLAG-NOD1 in three independent single cell clones (NF1, NF2 and NF3). (I) qPCR for the indicated genes in THP-1 NOD1 cells following CRISPR/Cas9 mediated ablation of RIPK2 in three independent single cell clones. NT: non-targeting gRNA. NF: gRNA targeting FLAG-NOD1. RIPK2: gRNA targeting RIPK2. Data in (A) to (I) are representative of three independent experiments. Error bars on graphs are mean±SEM of at least three biological replicates and where not visible in (E) are shorter than the height of the symbol. p values from an unpaired t test (two-tailed) without assuming a consistent standard deviation are shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: For CRISPR/Cas9 targeting of RIPK2 or FLAG NOD1 , the guide RNA targeting RIPK2 or FLAG NOD1 (IDT) was cloned into a single self-inactivating lentivirus plasmid pRRL-U6-empty-gRNA-MND-Cas9-t2A-Puro, that expresses a Cas9-T2A-puromycin resistance cassette controlled by an MND promoter ( 45 ).

Techniques: Western Blot, Flow Cytometry, Expressing, CRISPR, Clone Assay, Two Tailed Test, Standard Deviation

Journal: Science signaling

Article Title: A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses

doi: 10.1126/scisignal.aba3244

Figure Lengend Snippet: (A) Histograms (left) and quantification (right) of NOD1 protein in THP-1 cells treated with scramble or miR-191 LNA or miR-15b/16 TSB. (B) qPCR for the indicated oncogenes in cells treated with miR-15b/16 TSB. Gene expression at each timepoint is represented relative to the scramble LNA-treated control at that timepoint (dashed line set to 1). (C) qPCR for miR-15b, miR-16, NOD1 and oncogenes in ex vivo human monocytes following inhibition of miR-15b and miR-16 with LNA. Gene expression is represented relative to that in the scramble LNA control (dashed line set to 1). (D) qPCR for miR-15b, miR-16 and NOD1 (left) and the indicated oncogenes (right) in THP-1 cells following CRISPR/Cas9 mediated reduction of miR15b/16 in two independent clones analyzed 4–6 weeks after miR-15b/16 targeting and single cell cloning. Gene expression is represented relative to that in the non-targeting (NT) gRNA control (dashed line set to 1). (E) qPCR for the indicated inflammatory genes in THP-1 cells following CRISPR/Cas9 reduction of miR15b/16. Data in A, B and D are representative of three independent experiments, and in E are representative of two independent experiments with three biological replicates per condition. In C, one representative of two independent donors is shown with three replicates per condition. Error bars on graphs are mean±SEM and p values from an unpaired t test (two-tailed) without assuming a consistent standard deviation are shown. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns: not significant.

Article Snippet: For CRISPR/Cas9 targeting of RIPK2 or FLAG NOD1 , the guide RNA targeting RIPK2 or FLAG NOD1 (IDT) was cloned into a single self-inactivating lentivirus plasmid pRRL-U6-empty-gRNA-MND-Cas9-t2A-Puro, that expresses a Cas9-T2A-puromycin resistance cassette controlled by an MND promoter ( 45 ).

Techniques: Expressing, Ex Vivo, Inhibition, CRISPR, Clone Assay, Two Tailed Test, Standard Deviation

Journal: Nucleic acids research

Article Title: CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

doi: 10.1093/nar/gku402

Figure Lengend Snippet: Figure 1. Schematic of CRISPR/Cas9 off-target sites with (A) 1-bp insertion (DNA bulge) or (B) 1-bp deletion (RNA bulge). The 20-nt guide sequence (orange line) in the sgRNA is shown with genomic target sequence (protospacer) containing single-base DNA bulge (red asterisk) or single-base sgRNA bulge (red ). The zoom-in sequences of protospacer and PAM are shown above the sgRNA guide sequence. Positions of nucleotides in the target are numbered 3′ to 5′ starting from the nucleotide next to PAM.

Article Snippet: CRISPR/Cas9 plasmid assembly DNA oligonucleotides containing a G followed by a 19- nt guide sequence (Supplementary Table S1) were kinased, annealed to create sticky ends and ligated into the pX330 plasmid that contains the +85 chimeric RNA under the U6 promoter and a Cas9 expression cassette under the CBh promoter (kindly provided by Dr Feng Zhang; it is also available at Addgene) (26).

Techniques: CRISPR, Sequencing

Journal: Nucleic acids research

Article Title: CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

doi: 10.1093/nar/gku402

Figure Lengend Snippet: Figure 4. Activity of sgRNA variants targeted to genomic loci containing single-base sgRNA bulges. (A and B) Activity of Cas9 at (A)HBB target site and (B)CCR5 target site carrying single-base sgRNA bulges associated with different variants of the original sgRNAs (A) R-01 and (B) R-30. Single nucleotide, A (green), G (black), C (blue), or U (red), was inserted into the original sgRNA throughout the guide sequence. Sequence of the original sgRNA is in the top row of the grid. Positions of the original guide sequence are shaded in gray, while the inserted positions are white. Due to identical nucleotides at adjacent positions, some inserted nucleotides can be in multiple positions (marked by ‘or’). Bar graphs on the right show corresponding cleavage activities quantified by T7E1 assay in HEK293T cells, with the same color scheme for different inserted nucleotides. Positions relative to PAM and the single nucleotides added are labeled on the y-axis. Error bar, SEM (n = 2).

Article Snippet: CRISPR/Cas9 plasmid assembly DNA oligonucleotides containing a G followed by a 19- nt guide sequence (Supplementary Table S1) were kinased, annealed to create sticky ends and ligated into the pX330 plasmid that contains the +85 chimeric RNA under the U6 promoter and a Cas9 expression cassette under the CBh promoter (kindly provided by Dr Feng Zhang; it is also available at Addgene) (26).

Techniques: Activity Assay, Sequencing, Labeling

Journal: Nucleic acids research

Article Title: CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

doi: 10.1093/nar/gku402

Figure Lengend Snippet: Figure 7. Paired Cas9 nickases with one bulge-containing sgRNA effectively cleave genomic DNA. (A) Human HBB gene targeted by Cas9 nickases (Cas9n) with paired guide strands R-01 and R-02. PAMs are indicated with grey bars. (B) T7E1 activities of Cas9n with R-01 bulge-variants paired with R-02, compared with original Cas9 activities of the R-01 bulge-variants as in Figures 2 and 4. Error bar, SEM (n = 2). Asterisks indicate P-values from a two-tailed independent two-sample t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: CRISPR/Cas9 plasmid assembly DNA oligonucleotides containing a G followed by a 19- nt guide sequence (Supplementary Table S1) were kinased, annealed to create sticky ends and ligated into the pX330 plasmid that contains the +85 chimeric RNA under the U6 promoter and a Cas9 expression cassette under the CBh promoter (kindly provided by Dr Feng Zhang; it is also available at Addgene) (26).

Techniques: Two Tailed Test

Journal: Nucleic acids research

Article Title: CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.

doi: 10.1093/nar/gku402

Figure Lengend Snippet: Figure 8. Activities of CRISPR/Cas9 nucleases at genomic target sites and at off-target sites with single-base DNA bulges coupled with mismatches. (A and B) On-target and off-target cleavage activities for (A) sgRNAs R-30 targeted to CCR5 gene, and (B) R-31 target to ERCC5 gene. Upper: target sequences (CCR5 and ERCC5) and off-target sequences (Off-4 and Off-1) with mismatch (red) and DNA bulge (shaded in yellow) shown next to the sgRNA (R-30 and R-31) tested. Red lines indicate the PAM. Bottom: Cleavage activities at the target sites and off-target sites measured by T7E1 assay in HEK293T cells. ‘−’ and ‘+’ denote samples treated without and with nuclease, respectively. Numbers below the lanes indicate average percentages of gene modification (n = 2). Asterisks indicate specific T7E1 cleavage products. (C and D) Sanger sequencing reads of amplified off-target sites aligned to the wild-type genomic sequence and sgRNAs for (C) R-30 and (D) R-31. The occurrence of each sequence is indicated to the left of the alignment, if greater than one. Unmodified reads are indicated by ‘WT’. Deletions are marked in gray and insertions marked in yellow. (E) Significant activities analyzed by deep sequencing at genomic off-target loci containing bulges coupled with mismatches and in some cases alternative NAG-PAM. Only bulge-containing off-target loci determined to have P-values less than 0.05 are shown. Table on the left shows numbers of mismatches at off-target loci in addition to bulge (no. of mis), bulge types, positions of bulges from PAM (bulge pos), labels for the loci as in Supplementary Table S6 and sequences of off-target sites including PAMs. In these off-target genomic sequences, mismatches are marked by red, deleted base compared to sgRNA marked as ‘−’ (sgRNA bulge), inserted base compared to sgRNA marked as underlined red letters (DNA bulge), NAG-PAMs are marked by blue. Bar graph on the right indicates indel percentages quantified for mock (blue) and treated samples (red) with sgRNAs at off-target loci shown in the table to the left. Error bars, Wilson intervals (see ‘Materials and Methods’ section). *P ≤0.05, ***P ≤0.001 as determined by Fisher’s exact test. The % indel values of treated samples are also indicated.

Article Snippet: CRISPR/Cas9 plasmid assembly DNA oligonucleotides containing a G followed by a 19- nt guide sequence (Supplementary Table S1) were kinased, annealed to create sticky ends and ligated into the pX330 plasmid that contains the +85 chimeric RNA under the U6 promoter and a Cas9 expression cassette under the CBh promoter (kindly provided by Dr Feng Zhang; it is also available at Addgene) (26).

Techniques: CRISPR, Modification, Sequencing, Amplification, Genomic Sequencing