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