System setup and MD simulations

1. The crystal structure of Cas9/R-loop complex captured in the precleavage state (PDB ID: 5F9R) was selected as the starting model for the simulations performed in this study.

2. The Mg²⁺ ions were treated with the ion model developed by Liao el al. (DOI: 10.1021/acs.jpclett.7b02358) and the midC4 parameter set was considered here. The TIP4PEw water model was used to describe the water molecules, as the above Mg²⁺ ion parameters were developed against this solvent model. 

3. Additionally, the following files (Mg_midC4.frcmod and Mg.lib) are needed to be read while generating the parameter and topology files for MD simulations via the AmberTools tleap:

###############

........

loadoff                    Mg.lib
loadAmberParams Mg_minC4.frcmod

........
###############

The content in Mg_minC4.frcmod:

###############

Mg2+ dummy model

MASS
MG  6.305
Da  3.000       
Db  3.000
Dc  3.000

BOND
MG-Da   800.0   0.900   
MG-Db   800.0   0.900
MG-Dc   800.0   0.900

ANGLE
Da-MG-Da     125.000   180.000
Db-MG-Db     125.000   180.000
Dc-MG-Dc     125.000   180.000
Da-MG-Db     125.000    90.000
Da-MG-Dc     125.000    90.000
Db-MG-Dc     125.000    90.000

DIHE

IMPROPER

NONBON
 MG   0.706    26.8375
 Da   0.000    0.0000 
 Db   0.000    0.0000
 Dc   0.000    0.0000
###############

The content in Mg.lib:

###############

!!index array str
"DMG"
!entry.DMG.unit.atoms table  str name  str type  int typex  int resx  int flags  int seq  int elmnt  dbl chg
"MG"  "MG" 0 1 131072 1 12 -1.000000
"DM1" "Da" 0 1 131072 2  1 0.500000
"DM2" "Db" 0 1 131072 3  1 0.500000
"DM3" "Dc" 0 1 131072 4  1 0.500000
"DM4" "Db" 0 1 131072 5  1 0.500000
"DM5" "Dc" 0 1 131072 6  1 0.500000
"DM6" "Da" 0 1 131072 7  1 0.500000
!entry.DMG.unit.atomspertinfo table  str pname  str ptype  int ptypex  int pelmnt  dbl pchg
"MG"  "MG" 0  12 0.0
"DM1" "Da" 0   1 0.0
"DM2" "Db" 0   1 0.0
"DM3" "Dc" 0   1 0.0
"DM4" "Db" 0   1 0.0
"DM5" "Dc" 0   1 0.0
"DM6" "Da" 0   1 0.0
!entry.DMG.unit.boundbox array dbl
-1.000000
0.0
0.0
0.0
0.0
!entry.DMG.unit.childsequence single int
2
!entry.DMG.unit.connect array int
0
0
!entry.DMG.unit.connectivity table  int atom1x  int atom2x  int flags
1 2 1
1 3 1
1 4 1
1 5 1
1 6 1
1 7 1
!entry.DMG.unit.hierarchy table  str abovetype  int abovex  str belowtype  int belowx
"U" 0 "R" 1
"R" 1 "A" 1
"R" 1 "A" 2
"R" 1 "A" 3
"R" 1 "A" 4
"R" 1 "A" 5
"R" 1 "A" 6
"R" 1 "A" 7
!entry.DMG.unit.name single str
"DMG"
!entry.DMG.unit.positions table  dbl x  dbl y  dbl z
 0.000  0.000  0.000
 0.000  0.000  0.800
 1.000  0.000  0.000
 0.000  1.000  0.000
-1.000  0.000  0.000
 0.000 -1.000  0.000
 0.000  0.000 -0.800
!entry.DMG.unit.residueconnect table  int c1x  int c2x  int c3x  int c4x  int c5x  int c6x
0 0 0 0 0 0
!entry.DMG.unit.residues table  str name  int seq  int childseq  int startatomx  str restype  int imagingx
"DMG" 1 8 1 "?" 0
!entry.DMG.unit.residuesPdbSequenceNumber array int
0
!entry.DMG.unit.solventcap array dbl
-1.000000
0.0
0.0
0.0
0.0
!entry.DMG.unit.velocities table  dbl x  dbl y  dbl z
0.0 0.0 0.0
0.0 0.0 0.0
0.0 0.0 0.0
0.0 0.0 0.0
0.0 0.0 0.0
0.0 0.0 0.0
0.0 0.0 0.0
###############

3. And use parmed to modify the system prmtop file (named complex_wat.prmtop here, for example) derived above to include the midC4 parameter for Mg2+ ions:

parmed -i parmed.in 

parmed.in contains the relevant settings read by parmed:

##########################################

parm          complex_wat.prmtop
loadRestrt  complex_wat.inpcrd
setOverwrite True

add12_6_4 @MG  c4file minC4file.dat

outparm complex_wat_Liao.prmtop complex_wat_Liao.inpcrd
##########################################

The content in minC4file.dat:

###############

Mg-1 67.0

###############

Note: remember to set "lj1264=1" in the MD configuration file to make the Liao Mg2+ ion model effective in MD simulations! 

4. The Mg²⁺ ions were docked at the interface between the RuvC nuclease center and the non-target DNA by referencing the crystal structure of a bacterial RNase H with an RNA/DNA hybrid (DOI: 10.1016/j.cell.2005.04.024).  

5. The built system (wild type Cas9 with a NGG PAM) underwent stages of energy minimization (2000 steps), slow heating (over 50 ps), equilibration (over 50 ns) to reach stable. A typical configuration file for ensuing production simulation is provided below.

######################

&cntrl
imin=0, 
nstlim=50000000,
dt=0.002,
nrespa=1,

irest=1,
ntx=5, 
ntxo=2,
ntpr=25000,
ntwr=25000,
ntwx=25000,
iwrap=1,
ioutfm=1,
ntwprt=0,
 lj1264=1,

ntt=3,
temp0=303.15,
gamma_ln=1,
ig=-1,

ntp=1,
barostat=2,
pres0=1.01325,
taup=2,

ntc=2,
ntf=2,
ntb=2,
cut=9,
/
######################

6. Building the mutated PAM systems was facilitated by VMD and tleap. In building the NAG system, for example, delete the 2nd G base atoms in the NGG PAM first, re-name the original G as A, and then use tleap to complete the missing base atoms of A. In VMD, adjust the orientation of A to avoid potential steric clashes if present. The generation of NNGG system followed the same strategy.