Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo

doi: 10.1002/dvdy.24586

Figure Lengend Snippet: A, A Model for Cas9 and Cas9-DA-mediated gene editing. CRISPR/Cas9 alters the genome through Non Homologous End Joining (NHEJ), whereas CRISPR/Cas9-DA is designed to edit specific C to T conversions on one DNA strand through deamination. The complementary G will be then converted to an A (labeled in blue) during replication or through the internal mis-matching repair mechanism in the subsequent cell cycle. Not all changes may be retained due to this repair. B, A schematic depicting normal sea urchin development. The embryo hatches at Day1, gastrulates, and starts forming skeletons at Day2 and reaches the larval stage with extended skeletons at Day3. C, Cas9 (top), sCas9-DA (middle), and dCas9-DA (bottom) construct designs. sCas9-DA has a mutation (D10A) in one of the nickase sites, whereas dCas9-DA has double mutations (D10A and H840A) to inactivate both nickase sites. Each construct possesses a SP6 promoter for IVT. Flag, Flag-tag; NLS, Nuclear Localization Signal.

Article Snippet: Preparation of Cas9 and Cas9-Deaminase Constructs The Cas9 construct was obtained from Addgene (plasmid #51307), which has been previously reported to function in Xenopus tropicalis ( Nakayama et al., 2013 ).

Techniques: CRISPR, Non-Homologous End Joining, Labeling, Construct, Mutagenesis, FLAG-tag

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo

doi: 10.1002/dvdy.24586

Figure Lengend Snippet: CRISPR/Cas9/Deaminase targeting of Alx1 evaluated at 3 dpf. A–B′, Proportion of injected individuals with skeleton formation (A and A′) and with each developmental stage (B and B′) at 3 dpf. n= total number of embryos/larvae observed. A′ and B′, Representative images of Cas9+Alx1 sgRNAs introduced embryos that show each phenotype used for scoring in A and B. A′, Embryos/larvae were scored in A for formation of skeletal rods, skeletal granule, or no-skeleton. B′, Embryos/larvae were scored in B for extended skeletal rod formation and elongated body shape as “larva”, for archenteron appearance with no extended skeletal rod as “gastrula”, and for blastocoel presence with no archenteron formation as “blastula”. Inhibition of full skeleton formation typically correlated with halted development and embryos were scored as gastrula. C-E, 3 dpf embryos/larvae injected with Cas9 (C), sCas9-DA (D) or dCas9-DA (E) with Alx1 sgRNAs. Membrane mCherry mRNA (red) was used as an injection marker. Brightfield was used with a polarizing-filter to identify skeleton formation (arrows). Arrowheads indicate archenteron. Cas9/sCas9/dCas9-only embryos/larvae were injected without sgRNAs as controls. Scale bars = 50um.

Article Snippet: Preparation of Cas9 and Cas9-Deaminase Constructs The Cas9 construct was obtained from Addgene (plasmid #51307), which has been previously reported to function in Xenopus tropicalis ( Nakayama et al., 2013 ).

Techniques: CRISPR, Injection, Inhibition, Membrane, Marker

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo

doi: 10.1002/dvdy.24586

Figure Lengend Snippet: CRISPR/Cas9/Deaminase targeting Dsh evaluated at 3 dpf. A & B, Proportion of injected individuals with skeleton formation (A) and with each developmental stage (B) at 3 dpf. n= total number of embryos/larvae observed. Inhibition of gastrulation (archenteron formation) results in delay of overall development. C & D, 3 dpf embryos/larvae injected at fertilization with Cas9 (C), or sCas9-DA (D) with Dsh sgRNAs. Membrane mCherry mRNA (red) was used as an injection marker. Brightfield was used with a polarizing-filter to identify skeleton formation (arrows). Arrowheads indicate archenteron. Cas9/sCas9-only embryos/larvae were injected without sgRNAs as controls. Scale bars = 50um.

Article Snippet: Preparation of Cas9 and Cas9-Deaminase Constructs The Cas9 construct was obtained from Addgene (plasmid #51307), which has been previously reported to function in Xenopus tropicalis ( Nakayama et al., 2013 ).

Techniques: CRISPR, Injection, Inhibition, Membrane, Marker

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo

doi: 10.1002/dvdy.24586

Figure Lengend Snippet: Sequencing results of Alx1-gRNA2 (A) and Dsh-gRNA2 (B) target and flanking sequences (~200bp) were amplified by PCR from the extracted genome of each single embryo/larva, and the PCR products were subcloned and subjected to individual sequencing. ~80bp of each PCR product is shown in this figure. The original gRNA target sequence is indicated in red, the PAM target within the genome in green, mutated nucleotides in blue, and deleted sequences with a dashed line. Blue boxes indicate representative target cytosine mutated by Cas9-DA. Yellow boxes indicate cytosine that could induce a stop codon when converted to thymidine, while no mutations were found in those regions. # of samples (%) indicates number and proportion of each mutational pattern. * The deletion and C-to-T conversion formed a stop codon (TAA) at the squared region. ** The deletion formed a stop codon (TAA) at 70bp downstream from the PAM sequence.

Article Snippet: Preparation of Cas9 and Cas9-Deaminase Constructs The Cas9 construct was obtained from Addgene (plasmid #51307), which has been previously reported to function in Xenopus tropicalis ( Nakayama et al., 2013 ).

Techniques: Sequencing, Amplification

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo

doi: 10.1002/dvdy.24586

Figure Lengend Snippet: sgRNA efficiency and consensus test targeting the polyketide synthase (PKS) gene. A, A schematic diagram of the PKS 3rd exon showing the 16 gRNA target sites. gRNAs #6 and #10 were previously used (highlighted in blue), and shown to cause an albino phenotype when introduced with Cas9 mRNA (Oulhen and Wessel, 2016). Primers (indicated in blue) for the genomic PCR were designed at the end of 5′ and 3′ ends, respectively. B–D, Three resulting phenotypic categories of the PKS mutation. (B) Captured images of 3 dpf larvae injected with Cas9-DA mRNA and 16 PKS sgRNAs. Larvae with multiple pigmented cells (arrows) were counted as “Normal pigment”, followed by “Reduced pigment”, and “No pigment” groups as shown in graph C. The insert at the bottom corner of each image is a magnified view of each squared region. (C) Graph showing average group allocations (normal pigment, reduced pigment, no pigment) between injection groups. The results are the average of two independent experiments. n= total number observed. (D) A magnified view of the arms of pluteus larvae. Pigment cells in the dCas9-DA group were often pale in color compared the control (arrows). E, Sequencing results of the PKS 3rd exon. Since the primer for the Region1 was designed within a close proximity to the gRNA#1 target site, only the Region 2 was analyzed for sequencing to avoid any confusion. Original Region2 sequence is indicated with the black bar; each gRNA site is indicated with arrows directing toward each PAM sequence; matched/unedited sequence of each clone from the Cas9 group is shown in red bar. Blank spaces of each clone indicate a deletion in the sequence. The diagram was constructed by SnapGene software. Mutations found in the sCas9-DA and dCas9-DA groups are summarized underneath each gRNA site. For details regarding the mutations, please refer to Tables. S5–S7. F, Sequencing results of the gRNA#15 target site of PKS exon3 sequence. gRNA#15 target site is shown in red, PAM in green, mutations in blue. The C->T and G-A mutations (black squares) formed a stop codon in each mutated clone. # of samples (%) indicates number and proportion of mutation patterns. Due to the prevalence of polymorphisms in these wild-type animals, it should be noted that the original PKS (SPU#002895) sequence was modified based on the sequencing result of the control (gRNA only) group.

Article Snippet: Preparation of Cas9 and Cas9-Deaminase Constructs The Cas9 construct was obtained from Addgene (plasmid #51307), which has been previously reported to function in Xenopus tropicalis ( Nakayama et al., 2013 ).

Techniques: Mutagenesis, Injection, Control, Sequencing, Construct, Software, Modification

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Purified SARS-CoV-2 RdRp proteins expressed in baculovirus-infected insect cells ( Spodoptera frugiperda , Sf) analysed by SDS–PAGE and Coomassie staining. 7H8: nsp7-His 6 -nsp8, nsp12-HF: nsp12-His 6 -3xFlag, nsp12-F/7H8: nsp12-3xFlag/nsp7-His 6 -nsp8, nsp12-HF/7L8: nsp12-His 6 -3xFlag/nsp7-GGSGGS-nsp8, nsp12-HF/7/8: nsp12-His 6 -3xFlag/nsp7/nsp8. ( B ) Bacterially expressed and purified SARS-CoV-2 nsp7, nsp8 and nsp12 proteins analysed by SDS–PAGE and Coomassie staining. The proteins were expressed as 14His-SUMO fusion proteins in E. coli . 14His-SUMO was removed by a SUMO-specific protease during purification generating native N-termini. ( C ) Gel-based primer-extension assay to test RNA-dependent RNA synthesis using the RdRp preparation Sf nsp12-F/7H8. The substrate consists of a 10 nt RNA primer annealed to the 3′ end of a 35 nt RNA template. The 5′ end of the template strand is labelled with a Cy3 fluorophore. Reaction products were analysed by native PAGE and visualisation of Cy3 fluorescence. Formation of duplex RNA by RdRp was observed over time. Controls: a preformed Cy3-labelled dsRNA with the same size as the reaction product (dsRNA), the primed substrate (no protein). ( D ) Schematic diagram illustrating the FRET-based RdRp strand displacement assay. The RNA substrate is composed of a Cy3 fluorophore-containing template strand, an annealed primer and an annealed quencher strand with a 5′ flap. RdRp activity synthesises RNA by extending the primer strand and displaces the quencher strand. The displaced quencher strand can no longer anneal to fully synthesised duplex RNA leading to an increase in Cy3 fluorescent signal. ( E ) FRET-based strand displacement assay using the indicated concentrations of Sf nsp12-F/7H8.

Article Snippet: Nsp12 was subcloned into pBIG1a(polh) either to contain a C-terminal His 6 -3xFlag tag (protein sequence: M-nsp12-GGSHHHHHHGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-His 6 -3xFlag, Addgene ID: 169182) or to contain a C-terminal 3xFlag tag (protein sequence: M-nsp12-GGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-3xFlag).

Techniques: Purification, Infection, SDS Page, Staining, Primer Extension Assay, Clear Native PAGE, Fluorescence, Activity Assay

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: RdRp was mixed at the indicated concentrations with 100 nM RNA substrate and 300 µM of each NTP and Cy3 fluorescence was recorded. Enzyme preincubation was performed where indicated at 22°C for 30 min at the indicated ratios (5 µM nsp12). ( A ) Sf nsp12-F/7H8. ( B ) Sf nsp12-HF/7L8. ( C ) Sf nsp12-HF after preincubation with Sf 7H8 (1 : 3 ratio). ( D ) Ec nsp12 after preincubation with Ec nsp7 and Ec nsp8 (1 : 3 : 6 ratio). ( E ) Ec nsp12 after preincubation with Sf 7H8 (1 : 3 ratio). ( F ) Sf nsp12-HF after preincubation with Ec nsp7 and Ec nsp8 (1 : 3 : 6 ratio).

Article Snippet: Nsp12 was subcloned into pBIG1a(polh) either to contain a C-terminal His 6 -3xFlag tag (protein sequence: M-nsp12-GGSHHHHHHGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-His 6 -3xFlag, Addgene ID: 169182) or to contain a C-terminal 3xFlag tag (protein sequence: M-nsp12-GGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-3xFlag).

Techniques: Fluorescence

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A – C ) Strand displacement assay using insect cell expressed (Sf) nsp12-HF/7L8 at the indicated concentrations alone ( A ) or after pre-incubating at 22°C for 30 min with Ec nsp7 and Ec nsp8 (ratio 1 : 3 : 6) ( B ) or with Sf 7H8 (ratio 1 : 6) ( C ). As control, a condition without NTPs using 200 nM RdRp was included. ( D ) Optimisation experiment to decide on RdRp concentration using HTS conditions (100 nM strand displacement substrate, 300 µM NTPs). Sf nsp12-HF/7L8 was preincubated at 22°C for 30 min with Sf 7H8 at either 1 : 3 or 1 : 5 ratio as specified and tested in the strand displacement assay at the indicated concentrations. Under these conditions, the unquenched Cy3 template alone had an average fluorescence value of 50 × 10 3 a.u. ( E ) Reaction velocities extracted from the curves shown in ( D ).

Article Snippet: Nsp12 was subcloned into pBIG1a(polh) either to contain a C-terminal His 6 -3xFlag tag (protein sequence: M-nsp12-GGSHHHHHHGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-His 6 -3xFlag, Addgene ID: 169182) or to contain a C-terminal 3xFlag tag (protein sequence: M-nsp12-GGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-3xFlag).

Techniques: Control, Concentration Assay, Fluorescence

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Logistics of the screen. A custom chemical library consisting of over 5000 compounds was screened against RdRp activity using the FRET-based strand displacement assay in a 384-well format. RdRp was prepared by preincubation of Sf nsp12-HF/7L8 and Sf 7H8 in a 1 : 3 ratio for 30 min at room temperature. RdRp was dispensed into compound-containing 384-well plates and incubated for 10 min. Reactions were started by the addition of a substrate mix and florescence monitored in 90 s intervals. ( B,C ) Results of the HTS screen performed at 1.25 µM ( B ) and 3.75 µM ( C ) compound concentration. The normalised reaction velocity plotted against the compound number is shown. Validated hits are shown in red. ( D ) Kinetic curves with >15% reduction in reaction velocity or >10% reduction in fluorescent signal at the endpoint were inspected manually. As example, kinetic data for compound GSK-650394 is shown (red curve, data from surrounding wells in black). ( E ) Summary of the HTS hit selection strategy. From over 5000 compounds tested in the screen, 64 were considered primary hits after manual inspection of HTS reactions, which showed a reduction in reaction velocity below 85%, or a reduction in endpoint signal below 90%. Out of these, 46 primary hits were eliminated as they likely represent nonspecific modes of enzymatic inhibition such as colloidal aggregation or interference with the substrate structure. As part of this analysis promiscuous compounds that were identified as hits in other SARS-CoV-2 HTS [ , ] were removed with the exception of five suramin and suramin-like compounds, which were also identified in the SARS-CoV-2 nsp13 helicase HTS (Zeng et al. ). In vitro validation of the effect of suramin and suramin-like compounds on the activity of SARS-CoV-2 helicase and SARS-CoV-2 RdRp can be found in Zeng et al. . A total of 18 compounds (including five suramin and suramin-like compounds) were selected as hits, of which 14 were included in further in vitro validation in this work.

Article Snippet: Nsp12 was subcloned into pBIG1a(polh) either to contain a C-terminal His 6 -3xFlag tag (protein sequence: M-nsp12-GGSHHHHHHGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-His 6 -3xFlag, Addgene ID: 169182) or to contain a C-terminal 3xFlag tag (protein sequence: M-nsp12-GGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-3xFlag).

Techniques: Activity Assay, Incubation, Concentration Assay, Selection, Inhibition, In Vitro, Biomarker Discovery

Journal: Biochemical Journal

Article Title: Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

doi: 10.1042/BCJ20210200

Figure Lengend Snippet: ( A ) Concentration–response curves of selected compounds using the strand displacement assay. The experiment was performed using 150 nM RdRp, 100 nM RNA substrate and 300 µM of each NTP in the presence (+Triton) or absence (−Triton) of 0.01% Triton X-100. Quenching controls are shown in Supplementary Figure S5B. IC 50 values were calculated using Prism software. ( B,C ) Native gel-based assays using a primed RNA substrate as in . Reactions were started by mixing 300 nM RdRp complex (formed by preincubation of Sf nsp12-HF/7L8 and Sf 7H8 at 1 : 3 ratio) with 50 nM RNA substrate and 1 mM NTPs. Reaction products were analysed by native PAGE and visualisation of Cy3 fluorescence. ( B ) RdRp reactions were incubated for the indicated amounts of time. ( C ) Validation of selected compounds using 30-min RdRp reactions. RdRp complex was incubated with 50 µM of the indicated compounds for 10 min before reactions were started by substrate addition. Controls: a preformed dsRNA with the same size as the reaction product (dsRNA), the primed substrate (no protein). ( D ) Chemical structures of selected RdRp inhibitors.

Article Snippet: Nsp12 was subcloned into pBIG1a(polh) either to contain a C-terminal His 6 -3xFlag tag (protein sequence: M-nsp12-GGSHHHHHHGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-His 6 -3xFlag, Addgene ID: 169182) or to contain a C-terminal 3xFlag tag (protein sequence: M-nsp12-GGSDYKDHDGDYKDHDIDYKDDDDK, pBIG1a_nsp12-3xFlag).

Techniques: Concentration Assay, Software, Clear Native PAGE, Fluorescence, Incubation, Biomarker Discovery

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot of histone Kla levels on nucleosomes isolated from HEK-293T cells that were incubated with recombinant Sirt6 and/or NAD + . (B) Quantitation of the blot from panel A. Densitometry data were corrected based on the total protein stain then normalized to the untreated control condition. Groups were compared using a one-way ANOVA followed by Tukey’s post hoc test. n=3, error plotted as S.D. p (-/-v. +/+) = 0.006. (C) Western blot analysis of time courses of enzymatic deacylation reactions using 25 nM substrate nucleosome, 100 nM Sirt6, and 1 mM NAD + . (D) Quantitation of western blot data for the H3K9Ac substrate in panel C. V I was calculated for each substrate concentration by fitting the first three data points from this curve using standard linear regression. (E) As in panel D, but for the H3K9La substrate. (F) V I values plotted as a function of substrate concentration for each PTM substrate. For panels D, E, and F, n=3, error plotted as S.D.

Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.

Techniques: Western Blot, Isolation, Incubation, Recombinant, Quantitation Assay, Staining, Control, Concentration Assay

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot using a pan-lactyllysine antibody to analyze Kla levels on acid-extracted histones from wild type (“WT”), Sirt6 knockout (“S6KO”), and Sirt7 knockout (“S7KO”) U2OS cells in the presence of a titration of sodium L-lactate. Total protein was measured using a fluorescent total protein stain. (B) As in panel A but using a pan-acetyllysine antibody to analyze levels of histone Kac. (C) Western blots from panel A quantified by normalizing Kla signal to total protein then represented as a fold-change from the untreated condition. n=3, error plotted as S.D. Slopes of linear regressions were compared using Welch’s t-test. p (slope, S6KO v. WT) < 0.0001. p (slope, S7KO v. WT) = 0.39. (D) Western blots from panel B quantified by normalizing Kac signal to total protein then represented as a fold-change from the untreated condition. For WT and S6KO, n=3. For S7KO, n=2. Error plotted as S.D. (E) Baseline histone lactylation in untreated cells was measured using a pan-Kla antibody as in panel A and normalized to the loading control. The blot images used to generate this plot are shown in . Error plotted as S.D., p = 0.68.

Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.

Techniques: Western Blot, Knock-Out, Titration, Staining, Control

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot using a pan-lactyllysine antibody (PTM BIO) to analyze Kla levels on acid-extracted histones from wild type and Sirt6 knockout U2OS cells in the presence of a titration of rotenone, a mitochondrial Complex I inhibitor. Total protein was measured using a fluorescent total protein stain (LI-COR). (B) Antibody signal was corrected based on the loading control signal, then normalized as a fold-change from the untreated condition. n=3, error plotted as S.D. Slopes of linear regressions were compared using Welch’s t-test, p=0.0005 (C) As in panel A but using the pan-acetyllysine antibody to measure levels of histone Kac. (D) Quantitation of data from panel C. Data were processed as in panel B. n=3, error plotted as S.D.

Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.

Techniques: Western Blot, Knock-Out, Titration, Staining, Control, Quantitation Assay

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Levels of various histone acyl PTMs on acid-extracted histones from wild type or Sirt6 knockout U2OS cells were measured using site-specific antibodies (CST and PTM BIO). Total protein in each sample was measured using a fluorescent total protein stain (LI-COR). (B) Quantitation of data from panel A. Data were corrected based on the Revert 700 total protein stain, then normalized to the - Lactate control condition. Statistical analysis was performed using Student’s t-test and corrected for multiple hypothesis testing using the Holm-Šídák correction. n=3, error plotted as S.D. H3K9La p=0.013, H3K18La p=0.019.

Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.

Techniques: Knock-Out, Staining, Quantitation Assay, Control

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot measuring histone Kla on acid-extracted histones in WT and Sirt6-KO U2OS cell lines not treated with supplemental L-lactate with and without overexpression of human Sir6 under the control of a CMV promoter. WT histone H3 (CST) is used as a loading control. (B) Western blot measuring Sirt6 in the soluble protein fraction from cell lines from (a). β-Actin is used as a loading control. (C) Kla signal from (a) was quantified, normalized to the loading control, and represented as a fold change from the average of the WT condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (D) as in (a) but measuring histone Kac. (E) as in (c), but for Kac data from (d) (F) As in (a), but the cells were treated with 25 mM sodium L-lactate for 24 hours prior to histone extraction. (G) As in (b), but the cells were treated with 25 mM sodium L-lactate for 24 hours prior to cell lysis. (H) Kla signal from (f) was quantified, normalized to the loading control, and represented as a fold change from the average of the WT condition. (I) Inverse correlation of data from (f) and (g). Sirt6 signal was normalized by dividing by the β-actin loading control. Kla signal was normalized as in (h). The data were analyzed using a standard linear regression, R 2 = 0.84.

Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.

Techniques: Western Blot, Over Expression, Control, Extraction, Lysis

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot measuring histone Kla in WT and Sirt6-KO U2OS cells treated with sodium L-lactate and panobinostat, as indicated. A total protein stain (LI-COR) was used as a loading control. (B) As in panel A but measuring histone Kac. (C) Quantitation of selected data from panel A. Histone Kla signal was quantified using densitometry and corrected based on the total protein stain. Data are presented as a fold change from the untreated (-lactate, -panobinostat) condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p 1 = 0.03, p 2 = 0.02, p 3 =0.005, p 4 =0.002. n=3, error plotted as s.d. (D) Quantitation of selected data from panel B. Data was processed as in panel C. n=3, error plotted as s.d. Quantitation of additional conditions is available in figure S4.

Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.

Techniques: Western Blot, Staining, Control, Quantitation Assay

Journal: Frontiers in Plant Science

Article Title: Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

doi: 10.3389/fpls.2016.00743

Figure Lengend Snippet: Map of gene constructs. (A) pTRBO-prBChE-KDEL, (B) pTRBO-prBChE, and (C) p35S-P19, 35S: Cauliflower Mosaic Virus (CaMV) promotor, RAmy3DSP : rice alpha-amylase 3D gene signal peptide, Replicase : replicase gene of tobacco mosaic virus (TMV), MP : movement protein, BChE : codon optimized sequences for the human butyrylcholinesterase gene. 3XFLAG : specific amino acid codon sequences used for immunodetection and purification of the protein. KDEL : Codons encoding lysine, aspartic acid, glutamic acid, leucine, specifying the endoplamsmic retention sequence. P19: P19 gene from Tomato Bushy Stunt Virus (TBSV).

Article Snippet: The 3xFLAG tag sequence (Sigma–Aldrich, St. Louis, MO, USA) was inserted between the RAmy3D signal peptide and hBChE sequences in both cassettes.

Techniques: Construct, Immunodetection, Purification, Sequencing