Recombinant:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
DNA Extraction:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Cell Isolation:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Control:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Cell Culture:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Ligation:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Virus:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Crystallization Assay:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
Software:
Article Title: Structural basis for Cas9 off-target activity.
Article Snippet: Cas9 Nuclease V3 IDT Cat# 1081058 RoboSep Filter Tip Racks (8) StemCell Technologies Cat# 20125 Lonza Nucleofection Kits (10 96-well plates and buffer) P3 Lonza Walkersville, Inc. Cat# V4SP-3960 Recombinant Human IL-2 PeproTech, Inc Cat# 200-02 QuickExtract DNA Extraction Solution (10 mL Aliquots) Lucigen Cat# QE09050-PQ1329 RoboSep Buffer (5X Concentrate) StemCell Technologies Cat# 20124 RoboSep Kit # 17951 (Human T cell Isolation Kit) StemCell Technologies Cat# 17951 Dynabeads Human T-Activator CD3/CD28 (5 x 2 mL) Fisher Scientific Co LLC Cat# 11132D DNAse I Solution (1 mg/mL, 1 mL) StemCell Technologies Cat# 7900 Antibiotic/Antmycotic (6 x 100 mL) Fisher Scientific Co LLC Cat# MT30004CI CTS Immune Cell Serum Replacement (50 mL) ThermoFisher Scientific Cat# A2596101 Immunocult-XF T Cell Expansion Media StemCell Technologies Cat# 10981 SPRIselect Reagent Kit Beckman Coulter Cat# B233318 Dynabeads M-280 Streptavidin Invitrogen Cat# 11206D Q5 Hot Start High-Fidelty 2x Master Mix New England Biolabs Cat# M0494L NEBNext double-stranded DNA (dsDNA) Fragmentase New England Biolabs Cat# M0348L T4 DNA Ligase New England Biolabs Cat# M0202S 10x T4 DNA Ligase Reaction Buffer New England Biolabs Cat# B0202S Proteinase K ThermoFisher Scientific Cat# EO0491 HEPES Roth Cat# HN78.1 Magnesium chloride Fluka Cat# 63064-500G Potassium chloride Roth Cat# 6781.2 Potassium thiocyanate Roth Cat# P753.1 Poly(ethylene glycol) 3,350 Sigma-Aldrich Cat# 202444-500G Ethylenediaminetetraacetic acid Roth Cat# 8040.3 Ni Sepharose 6 Fast Flow resin (500 mL) Cytiva CAT# 17-5318-03 HiPrep 26/10 desalting columns (pack of 4) Cytiva CAT# 17-5087-02 5 mL HiTrap SP HP Cytiva CAT# 17-1152-01 HiLoad Superdex 200 26/600 Cytiva CAT# 28-9893-36 Critical commercial assays NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 MiSeq Reagent Kit v2 (300- cycles) Illumina, Inc Cat# MS-102- 2002 NextSeq PhiX Control Kit Illumina, Inc Cat# FC-110- 3002 Blood & Cell Culture DNA Maxi Kit Qiagen Cat# 13323 High Sensitivity DNA Bioanalyzer Kit Agilent Cat# 5067-4626 NEBNext dA-tailing Kit NEB Cat# E6053L NEBNext Ultra End Repair / dA-tailing module NEB Cat# E7442L NEBNext Ultra Ligation Kit NEB Cat# E7445L NextSeq 500/550 High Output V2.5 (150 cycles) Illumina Cat# 20024907 Deposited data Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 on-target DNA substrate Donohoue et al., 2021 PDB: 7OX9 Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #1 DNA substrate This study PDB: 7QQO (Continued on next page) e1 Cell 185, 4067–4081.e1–e6, October 27, 2022
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