Supplementary Information
Genome manipulation by guide
-
directed Argonaute cleavage
Shan Huang
1
*, Kaihang Wang
2
*, Stephen L. Mayo
1,2
*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 114
-
96,
1200 East California Boulevard, Pasadena, CA 91125, USA.
2
Division of Biology and Biological Engineering, California Institute of Technology, MC 147
-
75, 1200
East California Boulevard, Pasadena, CA 91125, USA.
* To whom correspondence should be addressed: gibbson783@gmail.com,
kaihangwang@caltech.edu,
smayo@caltech.edu
Present Address: Shan Huang, Department of Pediatrics, Division of Hematology, Oncology, Stem
Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford,
CA 94305, USA.
DNA sequence of the recombination cassette
.
Cyan: EM7 promoter, Yellow: kanamycin direct repeat sequence, Red: stop codon array,
Green:
kanamycin C
-
terminus sequence
CCACTAGTTAGCTCGAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAG
CATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGT
ATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGC
CCCTAACTCCGCCCAGTTCCGCCCATTC
TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAG
AGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT
AGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACG
TGTTGA
CAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACC
ATGA
GC
CATATTCAACGGGAAACGTCTT
GCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTA
TATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGG
GAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACA
GATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTAT
CCGTACTCCTGAT
TAACTGAC
TAG
CATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAA
TTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTG
CGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGG
TAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTC
TTCCGACCATCAAGCATT
TTATCCGTACTCCTGATGAT
GCATGGTTACTCACCACTGCGATCCCC
GGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCT
GGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCG
TATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGAT
GACGAGCGTAATGG
CTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTC
ACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATT
AATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTAT
GGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATA
ATCCTGAT
ATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTC
TAACACGTGCTACGAG
ATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGG
CTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATT
GCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCAC
TGC
ATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGAATTCCCGGGGATC
DNA sequence of the guide sequence
.
CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT
GCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC
CAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTG
CCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGC
A
GATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCC
GTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGC
TACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAAC
GGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTGAATTTGACCTGAGCGCATTT
T
TACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGG
AAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGAC
TACACAAATCAGCGATTTCCATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGG
AGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGC
A
GGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGG
TGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAA
ATCCCGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAG
AAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAA
Plasmid construction
pT
et_wtCas9 was a gift from Dr. Stanley Qi (Addgene plasmid #44250). pET28b_CbAgo and
pET28b_dCbAgo were gifts from Dr. Alexei Aravin.
pDL1999 was a gift from Dr. David Leach.
Plasmids pnonChikanS, pChikanS, p3×ChikanS, and p6×ChikanS were created to serve a
s
templates for PCR amplification to generate dsDNA donors for Lambda
-
Red recombineering.
Plasmid pnonChikanS was constructed as follows. Plasmid backbone consisting of two homology
arms to the cynX gene and an EM7 promoter was amplified from pDL1999 usin
g primers kan
-
bb.F/kan
-
bb.R. Two copies of kan gene were amplified from plasmid pET28b using primers
kan1.F/kan1.R and kan2.F/kan2.R, respectively. Each PCR product was gel
-
purified and all three
fragments were fused together by Gibson Assembly. In this wa
y, the recombination cassette
containing two non
-
functional copies of the kanamycin resistance gene separated by a stop codon
array was created and inserted adjacent to the EM7 promoter, between the two cynX homology arms.
Plasmid pChikanS was obtained thr
ough amplification of pnonChikanS with primers Chi.F/Chi.R.
PCR product was gel
-
purified and self
-
ligated by Gibson assembly.
Plasmid p3×ChikanS was obtained through amplification of pChikanS with primers
3×Chi.F/3×Chi.R. PCR product was gel
-
purified and s
elf
-
ligated by Gibson assembly.
Plasmid p6×ChikanS was obtained through amplification of p3×ChikanS with primers
6×Chi.F/6×Chi.R. PCR product was gel
-
purified and self
-
ligated by Gibson assembly.
To construct plasmids pTet_CbAgo and pTet_dCbAgo
, plasmid backbone was amplified from
pTet_wtCas9 using primers pTet
-
bb.F/pTet
-
bb.R. CbAgo and dCbAgo sequences were amplified from
pET28b_CbAgo and pET28b_dCbAgo respectively using primers Cb.F/Cb.R, gel
-
purified and ligated
with the plasmid backbone indi
vidually via Gibson Assembly.
To construct plasmids pTet_CbAgo/GS and pTet_dCbAgo/GS, a 1000
-
bp sequence homologous
to lacZ gene was amplified from DL1777 genomic DNA via colony PCR using primers GS
-
lacZ.F/GS
-
lacZ.R. Plasmid backbones of pTet_CbAgo and pTe
t_dCbAgo were also PCR amplified with primers
GS
-
bb.F/GS
-
bb.R, gel
-
purified and ligated with the 1000
-
bp lacZ homologous sequence individually
via Gibson Assembly.
To construct plasmids pTet_CpAgo/GS, pTet_CaAgo/GS, pTet_CdAgo/GS, pTet_IbAgo/GS,
plasmid ba
ckbones were amplified from pTet_CbAgo/GS using primers pTet
-
bb.F/pTet
-
bb.R. CpAgo,
CaAgo, CdAgo, IbAgo gene fragments were ordered from IDT (Integrated DNA Technologies) or
Twist Bioscience, amplified using primers pTet
-
Ago.F/pTet
-
Ago.R individually, gel
-
purified and ligated
with the plasmid backbones individually via Gibson Assembly.
To construct plasmids pEmpty and
pGS, pTet_CbAgo and pTet_CbAgo/GS were amplified using primers empty.F/empty.R respectively
to remove the CbAgo gene from the plasmid. The re
sulting PCR products were gel
-
purified and self
-
ligated via Gibson Assembly.
Plasmid pCas9
-
Red was purchased from Sigma
-
Aldrich (Catalog Number CAS9BAC1P), which
contains the gene for Cas9 from
Streptococcus pyogenes
(spCas9) expressed from its native
pro
moter, and the genes for Lambda
-
Red recombinases Exo, Beta, and Gam under control of the
arabinose
-
inducible ParaB promoter.
To construct plasmid pgRNA_cynX, plasmid pgRNA_lacZ was used as the template, which
encodes a CRISPR
-
gRNA targeting lacZ under cont
rol of the J23119 promoter and a sacB gene from
Bacillus subtilis
for counter
-
selection.
Plasmid backbone was amplified from
pgRNA_lacZ using
primers gRNA
-
cynX.F/gRNA
-
cynX.R, gel
-
purified, and self
-
ligated by Gibson Assembly, resulting in
the replacement o
f the original spacer sequence with the synthetic spacer sequence targeting cynX.
Strain construction
Strain DL1777, DL2859, and DL2874 were gifts from Dr. David Leach. SMR6669 was a gift from Dr.
Susan Rosenberg. SIJ488 was a gift from Dr. Alex Nielsen (A
ddgene bacterial strain #68246).
To generate strain
nonChikanS, ChikanS, 3×ChikanS, 6×ChikanS,
plasmid p
nonChikanS,
pChikanS, p3×ChikanS, and p6×ChikanS
were amplified with primers cynX
-
arm.F/cynX
-
arm.R
respectively and PCR products were gel
-
purified as dsDNA donors. DL1777
was transformed with
pCas9
-
Red, plated on LB plates supplemented with kanamycin, and incubated overnight at 30 °C.
One of the transformants was inoculated in 5 mL LB media supplemente
d with kanamycin and grown
at 30 °C until
OD600 = 0.3
-
0.4
. The Lambda
-
Red genes were then induced with 15 mM L
-
arabinose
for 45 min. The culture was used to prepare electrocompetent cells by washing twice with 10%
glycerol and resuspending in 50 μl 10% gly
cerol. 5
μ
l mixture of ~250 ng dsDNA and 100 ng plasmid
pgRNA_lacZ
was added to the cells, which were then subject to electroporation and allowed to
recover in 1 ml LB for 2 h at 30 °C. The recovered cells were plated on LB plates supplemented with
ampicil
lin and kanamycin and incubated overnight at 30 °C. Colonies with correct genomic integration
were verified by colony PCR and sequencing. Upon obtaining positive hits, plasmids were cured by
growing the cells overnight in LB media without antibiotics at 37
°C and plating on LB plates
supplemented with 5% sucrose. Successful plasmid curing was verified by colony PCR.
Strain 3×ChikanR was generated by previously described CbAgo/GS mediated recombination
using strain 3×ChikanS.
Kanamycin
-
resistant colonies wit
h correct recombination were verified by
colony PCR and sequencing. Plasmids were then cured by growing the cells overnight in LB media
without antibiotics at 37 °C and plating on LB plates. Successful plasmid curing was confirmed by the
cell sensitivity t
o ampicillin.
To generate strain
3×ChikanS_pal246 and 3×ChikanS_pal246_
∆
sbc
CD,
genomic DNA of
3×ChikanS was
amplified
via colony PCR
with primers cynX
-
arm.F/gmR
-
arm.R and PCR product was
gel
-
purified as dsDNA donor. DL2859 and DL2874
were individually tran
sformed with pCas9
-
Red,
plated on LB plates supplemented with kanamycin, and incubated overnight at 30 °C. One of the
transformants was inoculated in 5 mL LB media supplemented with kanamycin and grown at 30 °C
until
OD600 = 0.3
-
0.4
. The Lambda
-
Red genes w
ere then induced with 15 mM L
-
arabinose for 45 min.
The culture was used to prepare electrocompetent cells by washing twice with 10% glycerol and
resuspending in 50 μl 10% glycerol. 5
μ
l mixture of ~250 ng dsDNA and 100 ng plasmid
pgRNA_lacZ
was added to t
he cells, which were then subject to electroporation and allowed to recover in 1 ml LB
for 2 h at 30 °C. The recovered cells were plated on LB plates supplemented with ampicillin and
kanamycin and incubated overnight at 30 °C. Colonies with correct genomic
integration were verified
by colony PCR and sequencing. Upon obtaining positive hits, plasmids were cured by growing the
cells overnight in LB media without antibiotics at 37 °C and plating on LB plates supplemented with
5% sucrose. Successful plasmid cur
ing was verified by colony PCR.
To generate strain 3×ChikanS_
∆
rec
BCD and 3×ChikanS_
∆
recA
, chloramphenicol
-
resistance
cassettes were amplified from plasmid pDL1999 using primers recA.cmR.F/recA.cmR.R and
recBCD.cmR.F/recBCD.cmR.R respectively. PCR products
were gel
-
purified and used as dsDNA
donors. 3×ChikanS was transformed with pKD46 plasmid,
plated on LB plates supplemented with
ampicillin, and incubated overnight at 30 °C
. A 5 ml culture inoculated from single colony was grown
at 30°C until OD600 = 0.3
-
0
.4.
The Lambda
-
Red genes were then induced with 15 mM L
-
arabinose
for 45 min. The culture was used to prepare electrocompetent cells by washing twice with 10%
glycerol and resuspending in 50 μl 10% glycerol. These cells were transformed with ~250 ng dsDNA
by electroporation and allowed to recover in 1 mL LB for 2 h at 30 °C. The recovered cells were plated
on LB plates
supplemented with chloramphenicol and incubated overnight at
37 °C
. The correct
knockouts were verified by colony PCR and sequencing. Upon o
btaining positive hits, the pKD46
plasmid was cured by growing the cell in LB media at 37°C overnight and plating on LB plates.
Successful plasmid curing was confirmed by the cell sensitivity to ampicillin.
To generate strain SIJ488_
∆
recA
, the chlorampheni
col
-
resistance cassette was amplified from
plasmid pDL1999 using primers recA.cmR.F/recA.cmR.R. PCR products were gel
-
purified and used
as dsDNA donor. A 5 ml culture inoculated from single colony of SIJ488 was grown at 37°C until
OD600 = 0.3
-
0.4.
The Lamb
da
-
Red genes were then induced with 15 mM L
-
arabinose for 45 min.
The culture was used to prepare electrocompetent cells by washing twice with 10% glycerol and
resuspending in 50 μl 10% glycerol. These cells were transformed with ~250 ng dsDNA by
electropo
ration and allowed to recover in 1 mL LB for 2 h at 37 °C. The recovered cells were plated
on LB plates
supplemented with chloramphenicol and incubated overnight at
37 °C
. Correct
knockouts were verified by colony PCR and sequencing.
To generate
strain SIJ488_
∆
lacZ
, the kanamycin
-
resistance cassette was amplified from
genomic
DNA of
3×ChikanR via colony PCR
with primers lacZ.kanR.F/lacZ.kanR.R, and the PCR product was
gel
-
purified as dsDNA donor.
A 5 ml culture inoculated from single colony of SIJ
488 was grown at
37°C until OD600 = 0.3
-
0.4.
The Lambda
-
Red genes were then induced with 15 mM L
-
arabinose for
45 min. The culture was used to prepare electrocompetent cells by washing twice with 10% glycerol
and resuspending in 50 μl 10% glycerol. These c
ells were transformed with ~250 ng dsDNA by
electroporation and allowed to recover in 1 mL LB for 2 h at 37 °C. The recovered cells were plated
on LB plates
supplemented with kanamycin and incubated overnight at
37 °C
. Correct knockouts
were verified by co
lony PCR and sequencing.
Figure
S
1
.
Summary of
results
in Ref.
1
.
(i)
A
pre
-
installed
246
-
bp
palindrome
(pal246)
in the
chromosome leads to a
hairpin
formation
on the lagging strand during DNA replication
. (ii)
The hairpin
is recognized and cleaved by
E. coli
endogenous nuclease SbcCD
. (iii)
SbcCD cleavage on the
hairpin
generates
a two
-
ended DSB. (i
v
)
RecBCD binds to t
he DSB
and
starts
processing
chromosome DNA. (v) Recognition of Chi sites by RecBCD attenuates
its DNA degradation activity
and
initiate
s recombination between sister chromosomes
.
(vi)
Recombination results in
the deletion of
the direct repeat sequence.
In the original research, by removing one copy of direct repeat sequences
as well as the stop cod
on array between the two direct repeat sequences,
the recombination
generates
a functional zeocin resistance gene
in the host genome.
Figure
S
2
.
Possible mechanisms
for
CbAgo
gDNA biogenesis
. As found in the previous report,
CbAgo generates and
binds small gDNA from plasmids (
2
). Small DNA fragments can be generated
from guide
-
free CbAgo mediated plasmid degradation (“chopping” activity), likely at partially unwound
dsDNA in AT
-
rich region
s
(step i) (
3
).
The detailed
mechanism
of pAgo chopping
-
mediated gDNA
biogenesis
has been
illustrat
ed using TtAgo (
4
).
Alternatively, RecBCD
-
dependent plasmid
degradation during plasmid replication (step
s
ii and iii) can also generate small DNA debris, a process
similar to the spacer acquisiti
on in CRISPR adaptation (
5
-
7
). The resulting small DNA fragments can
serve as substrates for gDNA loading by CbAgo. The RecBCD
-
dependent plasmid degradation may
also be responsible for dCbAgo gDNA loading.
Figure
S
3
.
Effects of the number of Chi sites and GS length on the recombination frequency
. (A)
Recombination frequencies in different genetic contexts. Error bars, mean ± s.d. from three
independent
cultures. (B) Recombination
frequencies of strain 3×ChikanS expres
sing CbAgo in the
presence of GS with
varying
lengths. Error bars, mean ± s.d. from three independent cultures. n.s.
P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure
S
4
.
Genetic structure of engineered DSB. A pal246 was inserted
into lacZ loci, whose
cleavage by
E. coli
endogenous nuclease SbcCD forms DSB and stimulates
chromosome
recombination.
This
recombination
should undergo the
same
pathway shown in Figure S1.
Figure
S
5
.
Viabilities of cells in different genetic contexts. Error bars, mean ± s.d. from eight
independent cultures.
Table S1.
Strains used in this study.
Name
Description
Source
DL1777
MG1655
lacIq lacZχ
-
fnr
-
267 (∆ynaJ ∆ydaA ∆frn ∆ogt
∆abgT ∆abgB ∆abgA ∆
abgR ∆ydaL ∆ydaM ∆dboA
∆ydaO)
(
1
)
DL2859
DL1777
lacZ::pal246 cynX::Gm
R
(
1
)
DL2874
DL2859
∆
sbcCD
(
1
)
nonChikanS
DL1777
cynX
::[χ
-
kan cassette
]
This study
ChikanS
DL1777
cynX
::[χ
kan cassette
]
This study
3×ChikanS
DL1777
cynX
::[χχχ
kan cassette
]
This study
6×ChikanS
DL1777
cynX
::[χχχχχχ
kan cassette
]
This study
3×ChikanS_pal246
3×ChikanS
lacZ::pal246
This study
3×ChikanS_pal246_
∆
sbc
CD
3×ChikanS_pal246
∆
sbcCD
This study
3×ChikanS_
∆
recA
3×ChikanS
∆
recA::cm
R
This study
3×ChikanS_
∆
recBCD
3×ChikanS
∆
recBCD::cm
R
This study
3×ChikanR
DL1777
cynX
::
χχχ
ka
n
R
This study
SMR6669
MG1655 Δ
att
λ
::
psulA
-
gfpmut2
(
8
)
SIJ488
MG1655 Tn7
::
para
-
exo
-
beta
-
gam; prha
-
FLP; xylSpm
-
IsceI
Addgene
SIJ488_
∆
recA
SIJ488
∆
recA::cm
R
This study
SIJ488_
∆
lacZ
SIJ488
∆
lacZ
::ka
n
R
This study
Table S2.
Plasmids used in this study.
Name
Description
Source
pET28b
KmR; empty vector
Lab plasmid
collection
pET28b_CbAgo
KmR; pET28b
-
derived expression vector with N
-
terminally 6xHis
-
tagged
codon
-
optimized CbAgo
A. Aravin Lab
pET28b_dCbAgo
KmR; pET28b
-
derived expression vector with codon
-
optimized
N
-
terminally 6xHis
-
tagged catalytically dead CbAgo
A. Aravin Lab
pDL1999
CmR Ts SucS; pTOF24 +
cynX
::[
χ
-
zeo cassette
] fragment
(
1
)
pnonChikanS
CmR Ts SucS; pTOF24 +
cynX
::[
χ
-
kan cassette
] fragment
This study
pChikanS
CmR Ts SucS; pTOF24 +
cynX
::[
χ kan cassette
] fragment
This study
p3×ChikanS
CmR Ts SucS; pTOF24 +
cynX
::[
χχχ kan cassette
] fragment
This study
p6
×
ChikanS
CmR Ts
SucS; pTOF24 +
cynX
::[χχχχχχ
kan cassette
] fragment
This study
pCas9
-
Red
KmR Ts; encodes Cas9 from Streptococcus pyogenes (spCas9)
expressed from its native promoter, genes for λ
-
red
recombinases exo, beta, and gam under control of the arabinose
-
inducible
ParaB promoter
Sigma Aldrich
pgRNA_lacZ
AmpR SucS; encodes spCas9 gRNA targeting lacZ with a spacer
sequence 5'
-
CGGCCAGTGAATCCGTAATCA
-
3'
Lab plasmid
collection
pgRNA_cynX
AmpR SucS; encodes spCas9 gRNA targeting cynX
with a
spacer sequence 5'
-
CTCTGTGCAACCGGCTATTGC
-
3'
This study
pTet_wtCas9
AmpR; encodes
S. pyogenes
Cas9 under control of pTet
promoter
Addgene
pTet_CbAgo
AmpR; encodes CbAgo under control of pTet promoter
This study
pTet_dCbAgo
AmpR
; encodes dCbAgo under control of pTet promoter
This study
pTet_CbAgo
/GS
AmpR; encodes CbAgo under control of pTet promoter and
contains a 1000 bp sequence homologous to lacZ
This study
pTet_dCbAgo
/GS
AmpR; encodes dCbAgo under control of pTet promoter a
nd
This study
contains a 1000 bp sequence homologous to lacZ
pTet_CaAgo
/GS
AmpR; encodes CaAgo under control of pTet promoter and
contains a 1000 bp sequence homologous to lacZ
This study
pTet_CdAgo
/GS
AmpR; encodes CdAgo
under control of pTet promoter and
contains a 1000 bp sequence homologous to lacZ
This study
pTet_CpAgo
/
GS
AmpR; encodes CpAgo under control of pTet promoter and
contains a 1000 bp sequence homologous to lacZ
This study
pTet_IbAgo
/
GS
AmpR; encodes IbAgo
under control of pTet promoter and
contains a 1000 bp sequence homologous to lacZ
This study
pGS
AmpR; contains a 1000 bp sequence homologous to lacZ
This study
pEmpty
AmpR; empty vector
This study
pKD46
AmpR
Ts; encodes genes for Lambda
-
Red recombinases Exo,
Beta, and Gam under control of the arabinose
-
inducible ParaB
promoter
R. Phillips Lab
Table S3.
Oligo
nucleotide
s used in this study.
Name
Sequence 5'
-
3'
gRNA
-
cynX.F
GCAATAGCCGGTTGCACAGAGAGCTAGCATTATACCTAGGA
gRNA
-
cynX.R
TCTGTGCAACCGGCTATTGCGTTTTAGAGCTAGAAATAGCAAG
kan1.F
GACAAGGTGAGGAACTAAACCATGAGCCATATTCAACGGG
kan1.R
TTGAATATGCTAGTCAGTTAATCAGGAGTACGGATAAAATGC
kan2.F
TAACTGACTAGCATATTCAACGGGAAACGTC
kan2.R
TCGAAATCTCGTAGCACGTGTTAGAAAAACTCATCGAGCATC
kan
-
bb.F
CACGTGCTACGAGATTTCG
kan
-
bb.R
GGTTTAGTTCCTCACCTTGTC
Chi.F
CCCTGCACCACCAGCTAGCTCGAGGGTGTGGAAAGTCCCC
Chi.R
AGCTAGCTGGTGGTGCAGGGAATCGGTTTTATCATCGCCGGGC
3×Chi.F
GCGTGTCCACCAGCTCAGCATCGACCACCAGCCAGGCAGAAGTATGCAAAGC
3×Chi.R
TGCTGAGCTGGTGGACACGCGCTGGCTGGTGGTGCAGGGAATCGGTTTTATC
ATC
6×Chi.F
CGCCCAATGTCCCACCAGCTTGACCAATACCACCAGCCCCTAACTCCGCCCA
TCC
6×Chi.R
AGCTGGTGGGACATTGGGCGCTGGTGGACTGATGGCGGCTGGTGGTCGATG
CTGAG
cynX
-
arm.F
GTTATTGGCGCGGGTAGTATC
cynX
-
arm.R
TATCAAACACTCGCCTGGTG
gmR
-
arm.R
CGTGCGGTCATCACCTTAGATG
Cb.F
CTAAAGAGGAGAAAGGATCTATGGGGGGTTCTCATCATCATCATCATCATGG
Cb.R
CCTGGAGATCCTTACTCGAGTTATAAGAAGAACAAACGATTGTCTACTACACC
C
pTet
-
Ago.F
ATGACGATAAGGATCCGAGC
pTet
-
Ago.R
CCTGGAGATCCTTACTCGAG
pTet
-
bb.F
CTCGAGTAAGGATCTCCAGG
pTet
-
bb.R
AGATCCTTTCTCCTCTTTAGATC
GS
-
lacZ.F
AGCTCACTCAAAGGCGGTAACACTGGCCGTCGTTTTACAAC
GS
-
lacZ.R
TGATTCTGTGGATAACCGTATTTTCAATCCGCACCTCGCG
GS
-
bb.F
TACGGTTATCCACAGAATCAGGG
GS
-
bb.R
TTACCGCCTTTGAGTGAGCTG
empty.F
GGATCTATGTAACTCGAGTAAGGATCTCCAGGCATC
empty.R
GAGTTACATAGATCCTTTCTCCTCTTTAGATCTTTTG
recA.cmR.F
AAAAAGCAAAAGGGCCGCAGATGCGACCCTTGTGTATCAAACAAGACGATTAC
GCCCCGCCCTGCCACTC
recA.cmR.R
CAACAGAACATATTGACTATCCGGTATTACCCGGCATGACAGGAGTAAAACCG
GGAAGCCCTGGGCCAAC
recBCD.cmR.F
GTCGGATGCGACATGCGTAACACTCGTACGTCGCATCC
GGCAATTACGTTTAC
GCCCCGCCCTGCCACTC
recBCD.cmR.R
GCGAGATGACCCGCCTGCATTGCCCGAATCGTCAGTAGTCAGGAGCCGCTCC
GGGAAGCCCTGGGCCAAC
lacZ.kanR.F
GATTTCCTTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTATTAGAA
AAACTCATCGAGCATCAAATG
lacZ.kanR.R
GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCTGT
TGACAATTAATCATCGGC
Lambda.Red
.F
TAACCACTTCCAGCGCTGAG
Lambda.Red
.
R
TGGCCCTGCACGCGCCGTCG
Table S4.
The list of argonaute proteins used in this study.
Argonaute
GenBank
Organism
CaAgo
WP_042399050.1
Clostridium saudiense
CbAgo
WP_058142162.1
Clostridium butyricum
CdAgo
WP_055276084.1
Clostridium disporicum
CpAgo
EHP50500.1
Clostridium perfringens WAL
-
14572
IbAgo
WP_055087491.1
Intestinibacter bartlettii