KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Alt-R S.p. 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 × 2 mL)	Fisher Scientific Co LLC	Cat# 11132D
DNAse I Solution (1 mg/mL, 1 mL)	StemCell Technologies	Cat# 7900
Antibiotic/Antmycotic (6 × 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	This study	PDB: 7QQO
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #2 DNA substrate	This study	PDB: 7QR7
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #3 DNA substrate	This study	PDB: 7QQP
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #4 DNA substrate	This study	PDB: 7QQQ
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the AAVS1 off-target #5 DNA substrate	This study	PDB: 7QQR
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF on-target DNA substrate	This study	PDB: 7QQS
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #1 DNA substrate	This study	PDB: 7QQT
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #2 DNA substrate	This study	PDB: 7QQU
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #3 DNA substrate	This study	PDB: 7QQV
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #4 DNA substrate	This study	PDB: 7QQW
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #5 DNA substrate	This study	PDB: 7QQX
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #6 DNA substrate	This study	PDB: 7QR5
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the FANCF off-target #7 DNA substrate	This study	PDB: 7QQZ
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the PTPRC off-target #1 DNA substrate	This study	PDB: 7QR8
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the TRAC on-target DNA substrate	Donohoue et al., 2021	PDB: 7OX8
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the TRAC off-target #1 DNA substrate	This study	PDB: 7QR0
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the TRAC off-target #2 DNA substrate	This study	PDB: 7QR1
Atomic coordinates and structure factors of the ternary SpCas9 complex bound to the CD34 off-target #9 DNA substrate	This study	PDB: 7ZO1
Raw SITE-Seq data	This study	BioProject ID: PRJNA862989
Raw SITE-Seq data – TRAC target	Donohoue et al., 2021	BioProject ID: PRJNA744493
Bacterial and virus strains
E. coli Rosetta 2 (DE3)	Novagen	Cat# 71400–3
Oligonucleotides
Transcription templates, cleavage substrates, crystallization substrates	This study	Table S9
Recombinant DNA
pMJ806 (WT SpCas9)	AddGene	#39312
pMJ841 (SpCas9 D10A, H840A)	AddGene	#39318
Software and algorithms
XDS	Kabsch, 2010	http://xds.mpimf-heidelberg.mpg.de/
Phenix	Adams et al., 2010	http://www.phenix-online.org/
PyMOL	The PyMOL Molecular Graphics System, Version 2.0 Schrö dinger, LLC	https://www.pymol.org/
DynDom	Poornam et al., 2009	http://fizz.cmp.uea.ac.uk/dyndom/
PDBePISA	Krissinel and Henrick, 2007	http://www.ebi.ac.uk/pdbe/pisa/
ChimeraX	Pettersen et al., 2021	https://www.rbvi.ucsf.edu/chimerax/
GraphPad Prism 9	GraphPad Prism 9 for Windows, Version 9.1.2.	https://www.graphpad.com
MolProbity	Chen et al., 2010	http://molprobity.biochem.duke.edu
COOT	Emsley et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/