Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Recombinant, Transfection, Clone Assay, Protease Inhibitor, Software

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Comparative mRNA distribution of mouse Fam20A, Pcsk9 and Fam20C. Sections of whole body P10 WT mouse were subjected to IHyH using mFam20A, mPsck9, and mFam20C cRNA probes. (A) Representative sagittal cryostat sections of P10 mouse with thionin staining. (B-D) X-ray film autoradiography showing: (B) Fam20A mRNA distribution pattern restricted to few tissues including the molar, incisor, cerebellum, liver, kidney papilla and bone; (C) Pcsk9 mRNA distribution pattern restricted to the olfactory tract, molar, cerebellum, liver, stomach wall and small intestine wall; (D) Fam20C mRNA widespread distribution pattern with high levels in bone, molars, incisive, brain with thalamic structures, hippocampus and cerebellum, liver, adrenal gland, kidney cortex and papilla. Abbreviations: Ad – adrenal gland; B – bone; BM – bone marrow; Br – brain; Cb – cerebellum; H – heart; Hi – hippocampus; KCx – kidney cortex; Inc – incisive; Li – liver; LI – large intestine; Lu – lung; Mo – molar; OT – olfactory tract; P – papilla; SI – small intestine; SpC – spinal cord; St – stomach; Te – testis; Th – thymus; Tha – thalamus; UB – urinary bladder; V - vertebrae. (E-H) Adult mouse liver IHyH. (E) High resolution hepatocyte staining, dry film lower resolution IHyH Fam20A (F), mPcsk9 (G) and mFam20C (H). (I) QPCR analysis of mRNA distribution in adult liver mouse extracts and in indicated cell line models. QPCR was achieved using specific Fam20A and Fam20C primers. Data shown represent means ± SEM of at least three independent experiments.

Article Snippet: Fam20C Kinase assays with PCSK9 peptide substrates Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Staining, Autoradiography

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Fam20C/Fam20A enhance PCSK9 secretion and activity on the LDLR. (A) HepG2-Fam20C−/− cells were transfected with PCSK9, Fam20A, Fam20C-WT or Fam20C (D478A), a catalytically inactive form. After 18h of serum starvation the media were collected, and cells lysed and subjected to Western blot (WB) analysis with antibodies against the indicated proteins. Representative blots and quantitative densitometry analyses of PCSK9 and Fam20C media are shown. Data represent means of at least three independent experiments. **p<0.01 vs. lane 2, $p<0.05 and $$p<0.01 vs. lane 3, #p<0.05 and ##p<0.01 vs. lane 4, obtained from one-way ANOVA coupled to Tukey's multiple comparisons tests. (bottom panel) Densitometry analysis of total LDLR and the corresponding statistical analysis. Data represent means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, Tukey's multiple comparisons tests. iLDLR- ~110 kDa immature form of the LDLR that lacks O-glycosylation. (B) Media of HepG2-Fam20C−/− cells, expressing the indicated constructs were subjected to dephosphorylation by λ-phosphatase then analysed for PCSK9 by WB using anti-V5 antibody. (C) HepG2-Fam20C−/− cells were incubated for 18h with conditioned media (CM) from HEK293 cells expressing Fam20A along with PCSK9 and Fam20C-WT (media swap). The collected cell lysates and CM were analysed as in (A). Representative blots and corresponding densitometry analysis are shown. Data shown represent means of at least three independent experiments. **p<0.01, ***p<0.001 vs. PCSK9-/Fam20C-, ##p<0.01 vs. PCSK9+/Fam20C-, one-way ANOVA coupled to Tukey's multiple comparisons tests. (D) Confocal IF microscopy of cell surface LDLR in HepG2-PCSK9−/− cells incubated 18h with control or PCSK9 CM produced as in (C). np = non-phosphorylated, p = phosphorylated. Representative images are from at least three independent experiments. Scale bar, 15 μm.

Article Snippet: Fam20C Kinase assays with PCSK9 peptide substrates Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Activity Assay, Transfection, Western Blot, Glycoproteomics, Expressing, Construct, De-Phosphorylation Assay, Incubation, Microscopy, Control, Produced

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: In vitro and cellular validation of Fam20C-targets on PCSK9. (A) Schematic representation of potential Fam20C-Ser substrates (S-X-E motif) on hPCSK9-V5. Pro- Pro domain; Catalytic- Catalytic Domain; H- hinge region; CHRD- C-terminal Cys-His–rich domain. (B) In vitro Fam20C kinase assay measurements with the PCSK9 synthetic peptides as substrates and their specific activity values are indicated (top panel). The peptide sequences are presented and their predicted Ser-phosphosites are in bold and underlined, whereas natural mutations are in bold (bottom panel). The data were obtained from three independent experiments in duplicate. ****p<0.0001, obtained using one-way ANOVA coupled to Tukey's multiple comparisons tests. (C and D) Levels of non-phosphorylated and phosphorylated PCSK9 peptides spanning (C) Ser47 (S47EEDGLAEAPEHGTTATFHR) and (D) Ser688 (HLAQAS688QELQ) as measured by PAC-qMS assay in the culture medium of HepG2-Fam20C−/− cells (C), naive HepG2 cells with PCSK9 overexpression, and naive HepG2 cells with PCSK9 and Fam20C overexpression (D).

Article Snippet: Fam20C Kinase assays with PCSK9 peptide substrates Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: In Vitro, Biomarker Discovery, Kinase Assay, Activity Assay, Over Expression

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Characterization of Fam20C-mediated Ser-phosphorylation on PCSK9 activity. (A) HepG2-PCSK9−/− cells were transfected with V5-tagged control (empty vector) or PCSK9 constructs (WT, R46L, S668R and E670G) in the presence of Fam20C/Fam20A to maximise phosphorylation. After 18h, the cells were lysed and analysed by WB using antibodies against the indicated proteins. Representative blots (top panel) and densitometry quantification (bottom panel) are shown. Data represent means ± SEM of at least three independent experiments. *p<0.05, ***p<0.001, one-way ANOVA coupled to Tukey's multiple comparisons tests. (B) HepG2-PCSK9−/− cells were incubated for 18h with the CM of HEK293 cells expressing V5-tagged empty vector or PCSK9 constructs (WT, R46L, S668R and E670G) in the presence of Fam20C/Fam20A (media swap). The cells were then lysed and analysed as in (A). Representative blots and densitometry analysis are shown (top and bottom panels, respectively). Data represent means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, one-way ANOVA coupled to Tukey's multiple comparisons tests. (C and D) HepG2-PCSK9−/− cells were incubated for 18h with the CM of HEK293 cells expressing V5-tagged control or PCSK9-WT and mutated constructs (E670G, D374Y, S47A, S666A, S668A, S688A and S666A/S668A) in the absence (−) or presence (+) of Fam20C/Fam20A as indicated. The cells were then lysed and analysed as in (A). Representative blots (top panels in C, and D) and densitometry quantification (D and bottom panel in C) are shown. (C) Data represent means ± SEM of five independent experiments. *p<0.05, **p<0.01, ***p<0.001, obtained by two-way ANOVA coupled to Tukey's multiple comparisons tests. (D) Data represent means ± SEM of three independent experiments. *p<0.05, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests.

Article Snippet: Fam20C Kinase assays with PCSK9 peptide substrates Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Control, Plasmid Preparation, Construct, Incubation, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Effects of PCSK9-phosphosphomimetics on LDLR degradation. (A) HepG2-PCSK9−/− cells were incubated for 18h with CM from HEK293 cells expressing V5-tagged control, PCSK9-WT or mutated constructs (E670G, D374Y, S47A, S47D, S666A, S666E, S668A, S668E, S688A and S688D) in the presence of Fam20C/Fam20A. The cells were then lysed and analysed by WB using antibodies against the indicated proteins. Representative blots (top panels) and densitometry quantification (bottom panel) are shown. The data represent means ± SEM of at least three independent experiments. **p<0.01, ***p<0.001 and ****p<0.0001, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests. (B) Dil-LDL uptake. HepG2-PCSK9−/− cells were incubated for 20h with phosphorylated PCSK9 CM from HEK293 cells expressing V5-tagged control or PCSK9 constructs (WT, E670G, D374Y, S47A, S47D, S666A, S666E, S668A, S668E, S688A, S688D and S668R), in the presence of Fam20C/Fam20A. During the last 3h of incubation, Dil-LDL was added to the CM. The cells were then fixed and the Dil-LDL fluorescence quantified. The data represent means ± SEM of at least three independent experiments in quadruplicates. *p<0.05, **p<0.01 and ***p<0.001 vs. Control, #p<0.05 and ##p<0.01 vs. WT, $p<0.05, one-way ANOVA coupled to Tukey's multiple comparisons tests. (C) Confocal IF of cell surface LDLR in HepG2-PCSK9−/− cells incubated with phosphorylated PCSK9 CM from HEK293 cells expressing V5-tagged control or PCSK9-WT, and mutated constructs (E670G, D374Y, S47A, S47D, S666A, S666E, S668A, S668E, S688A, S688D and S668R), in the presence of Fam20C/Fam20A. After 18h of incubation the cells were fixed and processed for IF. Representative images are shown for each condition. Scale bar, 15 μm. (D) Quantifications were derived from analyses of 8 fields/condition/experiment. Data are averages ± SEM of two independent experiments. ***p<0.001 and ****p<0.0001, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests.

Article Snippet: Fam20C Kinase assays with PCSK9 peptide substrates Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Incubation, Expressing, Control, Construct, Fluorescence, Derivative Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Ser-Phosphorylation of PCSK9 by Fam20C-Kinase Enhances its Ability to Degrade the LDLR

doi: 10.1161/ATVBAHA.119.313247

Figure Lengend Snippet: Effect of PCSK9 pro-domain and Furin in PCSK9-mediated LDLR degradation. (A) Effect of PCSK9 Δ33-58 deletion. HEK293 cells were incubated for 18h with CM from HEK293 cells expressing V5-tagged control, PCSK9-WT or mutated constructs, D374Y, E670G, Δ33-58 and Δ33-58-E670G in the presence of Fam20A (non-phosphorylated, left panel) or Fam20C/Fam20A (phosphorylated, right panel). Cell lysates were analysed by WB using the indicated antibodies. Representative blots (top panels) and densitometry quantification (bottom panels) are shown. Data represent means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, obtained by one-way ANOVA coupled to Tukey's multiple comparisons tests. (B-C) Fam20C affects Furin-mediated PCSK9 cleavage. (B) HEK293 cells were transfected with V5-tagged PCSK9-WT or Fam20C/Fam20A. After 18h of serum starvation, PCSK9 media were collected and analyzed by WB. Representative blots of at least three independent experiments are shown. The migration position of the Furin-cleaved PCSK9 (PCSK9-Δ218) as well as the % cleavage by endogenous Furin are emphasized. (C) CM from CHO-FD11 Furin−/− cells transfected with the indicated constructs (i.e., PCSK9-V5, Furin-V5 and Fam20C-WT/Fam20A). Cell lysates and media were analysed by WB and representative blots of at least three independent experiments are shown. (D) HepG2-PCSK9−/− cells were incubated for 18h with the media produced in (C) and LDLR levels analysed by WB. Representative blots (top panels) and densitometry quantification (bottom panels) are shown. Data represent means ± SEM of at least three independent experiments. *p<0.05, ***p<0.001, ****p<0.0001, obtained by two-way ANOVA coupled to Tukey's multiple comparisons tests; ns- non-significant. (E) Relative PCSK9-LDLR binding assay. Microplates pre-coated with recombinant LDLR EGF-AB domain, which contains the PCSK9 binding site, were incubated for 18h with non-phosphorylated and phosphorylated PCSK9 CM obtained from HEK293 cells expressing V5-tagged PCSK9-WT in the absence or presence of Fam20C/Fam20A, respectively. The binding affinity of PCSK9 to LDLR was determined following analyses with 8 different concentrations per sample and the calculated relative EC50 values of WT phosphorylated PCSK9 (WT-P) were normalized to WT non-phosphorylated PCSK9 (WT-NP) as shown in (B). Data are averages ± SEM of two independent experiments. *p<0.05, obtained by unpaired two-tailed student t-test.

Article Snippet: Fam20C Kinase assays with PCSK9 peptide substrates Recombinant human Fam20C (63-C) was purified from insect cells as described in Xiao et. al. 39 β-casein and PCSK9 peptides were synthesized by GenScript.

Techniques: Incubation, Expressing, Control, Construct, Transfection, Migration, Produced, Binding Assay, Recombinant, Two Tailed Test

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: (A) Unrooted phylogenetic tree of meningococcal Cas9 orthologs. Groups 1 (blue), 2 (orange), and 3 (green) have PIDs with >98%, ~52%, and ~86% identity to Nme1Cas9, respectively. Three representative Cas9 orthologs (one from each group) (Nme1Cas9, Nme2Cas9 and Nme3Cas9) are indicated.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques:

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: (A) Nme2Cas9 genome editing of endogenous human sites in HEK293T cells following transient transfection of Nme2Cas9- and sgRNA-expressing plasmids. 40 sites were screened initially (Table S1); the 14 sites shown (selected to include representatives of varying editing efficiencies, as measured by TIDE) were then re-analyzed in triplicate. An Nme1Cas9 target site (with an N4GATT PAM) was used as a negative control.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Transfection, Expressing, Negative Control

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: (A) In vitro cleavage assay of Nme1Cas9 and Nme2Cas9 in the presence of five previously characterized anti-CRISPR proteins (10:1 ratio of Acr:Cas9). Top: Nme1Cas9 efficiently cleaves its target in the absence of an Acr or in the presence of a negative control Acr (AcrE2). All five type II-C Acr families inhibited Nme1Cas9, as expected. Bottom: Nme2Cas9 inhibition mirrors that of Nme1Cas9, except for the lack of inhibition by AcrIIC5Smu.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: In Vitro, Cleavage Assay, CRISPR, Negative Control, Inhibition

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: (A) Nme2Cas9 and SpyCas9 efficiencies vary based on the locus and target site. Dual sites (flanked by NGGNCC sequences) throughout the genome were selected for direct comparisons of editing by the two orthologs in HEK293T cells. Box-and-whisker plots indicate editing efficiencies at 28 dual sites by Nme2Cas9 and SpyCas9 (left). The sites that showed no editing were excluded from the analysis. Relative efficiencies of Nme2Cas9 and SpyCas9 show that Nme2Cas9 is less efficient than SpyCas9 (right), on average. Editing efficiencies by both Cas9 orthologs at all 28 sites were included in the analysis of relative efficiencies in the right panel.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Whisker Assay

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: (A) Workflow for delivery of AAV8.sgRNA.Nme2Cas9 to reduce serum cholesterol levels in mice by targeting Pcsk9. Top: schematic of the all-in-one AAV vector expressing Nme2Cas9 and the sgRNA. BGH, bovine growth hormone poly(A) site; HA, epitope tag; NLS, nuclear localization sequence; h, human-codon-optimized. Bottom: Timeline for AAV8.sgRNA.Nme2Cas9 tail-vein injections and analyses.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Plasmid Preparation, Expressing, Sequencing

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: (A) Workflow for single-AAV Nme2Cas9 editing ex vivo to generate albino C57BL/6NJ mice by targeting the Tyr gene. Zygotes are treated with AAV6.Nme2Cas9:sg Tyr and then transferred to the oviduct of pseudo-pregnant recipients.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Ex Vivo

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Virus, Recombinant, Transfection, Cloning, Protease Inhibitor, Software

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Recombinant, Transfection, Clone Assay, Protease Inhibitor, Software

Journal: Molecular cell

Article Title: A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing

doi: 10.1016/j.molcel.2018.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Abcam ab9485 Goat anti-PCSK9 R&D Systems AF3985 HRP-conjugated goat anti-rabbit Bio-Rad 1706515 Donkey anti-goat R&D Systems HAF109 Bacterial and Virus Strains Escherichia coli Rosetta (DE3) EMD Millipore Cat#70954 Escherichia coli DH5-alpha NEB Cat#C2987 Biological Samples Chemicals, Peptides, and Recombinant Proteins Ni-NTA agarose resin Qiagen Cat#30210 DNeasy blood and tissue kit Qiagen Cat#69504 DMEM ThermoFisher Cat#10566 Q5 High-fidelity PCR Master Mix NEB Cat#M0541X PolyFect Transfection reagent Qiagen Cat#301105 NEBuilder HiFi DNA assembly cloning kit NEB Cat#E5520 HiScribe T7 high yield RNA synthesis kit NEB Cat#E2040 cOmplete, Mini, EDTA-free protease inhibitor Sigma-Aldrich Cat#11836170001 T4 DNA ligase NEB Cat#M0202 T4 PNK NEB Cat#M0201 BfuAI NEB Cat#R0701 Critical Commercial Assays Deposited Data NCBI BioSample database SAMN09694825 Mendeley Data http://dx.doi.org/10.17632/tv8tswbd4b.1 Experimental Models: Cell Lines Human: HEK293T Human: K562 Mouse: Hepa1-6 Human: Primary Dermal Fibroblast; Normal, Adult (HDFa) ATCC PCS-201-012 Human: IMR90 ATCC CCL-186 Experimental Models: Organisms/Strains C57BL/6NJ Jackson Laboratory 005304 Oligonucleotides Listed in supplemental Table S1 IDT Recombinant DNA Codon-optimized Nme2Cas9 Genscript N/A Nme2Cas9 PID g-block IDT N/A Nme3Cas9 PID g-block IDT N/A Software and Algorithms TIDE Brinkman et al., 2014 FigTree http://tree.bio.ed.ac.uk/software/figtree/ Other Open in a separate window KEY RESOURCES TABLE Some meningococcal Cas9s have divergent PAM-interacting domains Nme2Cas9 is a compact Cas9 ortholog with a dinucleotide (N 4 CC) PAM requirement Nme2Cas9 edits mammalian genomes with little or no off-targeting Single-AAV delivery of Nme2Cas9 and its guide enables efficient editing in mice

Techniques: Recombinant, Transfection, Clone Assay, Protease Inhibitor, Software