KEY RESOURCE TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
HEPES	Sigma-Aldrich	H3375-250G
Sodium citrate tribasic dihydrate	Sigma-Aldrich	C8532-1KG
Citric acid	Sigma-Aldrich	C0759-500G
Sodium acetate	Sigma-Aldrich	S7545-1KG
Magnesium chloride hexahydrate	Sigma-Aldrich	M2393-500G
Potassium sulfate	Sigma-Aldrich	P9458-250G
Lithium chloride	Sigma-Aldrich	L9650-500G
glycerol	Sigma-Aldrich	G5516-1L
DL-Dithiothreitol (DTT)	Sigma-Aldrich	D9163-25G
Pentaerythritol propoxylate (5/4 PO/OH)	Sigma-Aldrich	41-8749-500ML
PEG 600	Sigma-Aldrich	202401-500G
PEG 3,350	Sigma-Aldrich	88276-1KG-F
PEG 6,000	Sigma-Aldrich	81255-1KG
T7 RNA polymerase	Home-made	N/A
Ulp1	Home-made	N/A
Toluidine Blue	AMRESCO	0672-25G
Urea	AMRESCO	0378-5KG
Spermidine	Sigma-Aldrich	S2626-1G
ATP	Sigma-Aldrich	A2383
UTP	Sigma-Aldrich	U6625
CTP	Sigma-Aldrich	C1506
GTP	Sigma-Aldrich	G8877
AxyPrep Plasmid Maxiprep Kit 25-prep	Axygen	AP-MX-P-25G
Critical Commercial Assays
Chelating Sepharose Fast Flow	GE Healthcare	Cat# 17-0575-02
HiTrap Heparin HP	GE Healthcare	Cat# 17-0407-03
HiTrap SP HP	GE Healthcare	Cat# 17-1152-01
Superdex 200 Increase, 10/300 GL	GE Healthcare	Cat# 28-9909-44
Deposited Data
The coordinates of Nme1Cas9-sgRNA complex	This paper	PDB: 6JDQ
The coordinates of Nme1Cas9 (His588Ala)-sgRNA-DNA in catalytic state	This paper	PDB: 6JDV
The coordinates of Nme1Cas9-sgRNA-DNA in seed-base paring state	This paper	PDB: 6KC7
The coordinates of Nme1Cas9(WT)-sgRNA-DNA in post-cleavage state	This paper	PDB: 6KC8
The coordinates of Nme2Cas9-sgRNA-dsDNA-0nt complex	This paper	PDB: 6JFU
The coordinates of Nme2Cas9-sgRNA-dsDNA-5nt complex	This paper	PDB: 6JE3
The coordinates of Nme1Cas9-sgRNA-AcrIIC3 complex	This paper	PDB: 6JE9
The coordinates of Nme1Cas9-sgRNA-AcrIIC3-dsDNA complex	This paper	PDB: 6JE4
The coordinates of SpyCas9-sgRNA-DNA complex	Anders et al., 2014.	PDB: 4UN3
The coordinates of SauCas9-sgRNA-DNA complex	Nishimasu et al., 2015.	PDB: 5CZZ
The coordinates of SpyCas9-sgRNA-DNA complex	Jiang et al., 2016.	PDB: 5F9R
Experimental Models: Organisms/Strains
Escherichia coli Rosetta (DE3)	Novagen	Cat# 70954-3
Recombinant DNA
pET28a-Nme1Cas9, various mutants	This paper	N/A
pET28a-Nme2Cas9, various mutants	This paper	N/A
pET28a-AcrIIC3	This paper	N/A
pET30b-Nme1Cas9	This paper	N/A
Oligonucleotides
147-nt SgRNA sequence. GGUCACUCUGCU AUUUAACUUUACGUUGUAGCUCCCUUUC UCAUUUCGGAAACGAAAUGAGAACCGUUG CUACAAUAAGGCCGUCUGAAAAGAUGUGC CGCAACGCUCUGCCCCUUAAAGCUCCUGC UUUAAGGGGCAUCGUUUAUC	This paper	N/A
135-nt SgRNA sequence. GGUCACUCUGCUAU UUAACUUUACGUUGUAGCUCCCUUUCUCG AAAGAGAACCGUUGCUACAAUAAGGCCGUC UGAAAAGAUGUGCCGCAACGCUCUGCCCC UUAAAGCUCCUGCUUUAAGGGGCAUCGU UUAUC	This paper	N/A
122-nt SgRNA sequence. GGUCACUCUUCUAU UUACCUAUACGUUGUAGCUCCCUUUCUCAUU UCGGAAACGAAAUGAGAACCGUUGCUACAAUA AGGCCGUCUGAAAAGAUGUGCCGCAACGCUC UUUCUAGAGCGUUC	This paper	N/A
102-nt SgRNA sequence. GGACUCACGACGCA UUACCUUUACGUUGUAGCUCCCUUUCUCAUU UCGGAAACGAAAUGAGAACCGUUGCUACAAUA AGGCCGUCUGAAAAGAUGUGCCAUC	This paper	N/A
SpyCas9 sgRNA sequence. GGAAAUUAGGUGCGC UUGGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGCUUC	This paper	N/A
SauCas9 sgRNA sequence. GGUCUGCUAUUUCUA UUUACGUUUUAGUACUCUGGAAACAGAAUCUAC UAAAACAAGGCAAAAUGCCGUGUUUAUC UCGUCAACUUGUUGGCGAGAUC	This paper	N/A
DNA sequences	This paper	Table S4
Plasmids and primers	This paper	Table S5
Software and Algorithms
PHENIX	Adams et al., 2002	http://www.phenix-online.org/
HKL2000	Otwinowski and Minor, 1997	http://www.hkl-xray.com/
COOT	Emsley et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
PyMOL	Molecular Graphics System, Version 1.8 Schrödinger, LLC	https://www.pymol.org/2/
PHASER	McCoy et al., 2007	https://www-structmed.cimr.cam.ac.uk/phaser_obsolete/
Other
Amicon Ultra Centrifugal Filters Ultracel-50K	Merck Millipore Ltd	UFC903096
Amicon Ultra Centrifugal Filters Ultracel-10K	Merck Millipore Ltd	UFC801096
3 Well Midi Crystallization Plate (Swissci)	HAMPTON RESEARCH	Cat# HR3-125