4078–4085
Nucleic Acids Research, 2023, Vol. 51, No. 8
Published online 17 March 2023
https://doi.org/10.1093/nar/gkad188
Genome manipulation by guide-directed Argonaute
cleavage
Shan Huang
1,*
, Kaihang Wang
2,*
and 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 and
2
Division of Biology and Biological Engineering, California
Institute of Technology, MC 114-96, 1200 East California Boulevard, Pasadena, CA 91125, USA
Received August 12, 2022; Revised February 24, 2023; Editorial Decision March 01, 2023; Accepted March 12, 2023
ABSTRACT
Many prokaryotic argonautes (pAgos) mediate DNA
interference by using small DNA guides to cleave tar-
get DNA. A recent study shows that CbAgo, a pAgo
from
Clostridium butyricum
, induces DNA interfer-
ence between homologous sequences and gener-
ates double-stranded breaks (DSBs) in target DNAs.
This mechanism enables the host to defend against
invading DNAs such as plasmids and viruses. How-
ever, whether such a CbAgo-mediated DNA cleavage
is mutagenic remains unexplored. Here we demon-
strate that CbAgo, directed by plasmid-encoded
guide sequences, can cleave genome target sites
and induce chromosome recombination between
downstream homologous sequences in
Escherichia
coli
. The recombination rate correlates well with
pAgo DNA cleavage activity and the mechanistic
study suggests the recombination involves DSBs
and RecBCD processing. In RecA-deficient
E. coli
strain, guide-directed CbAgo cleavage on chromo-
somes severely impairs cell growth, which can be
utilized as counter-selection to assist Lambda-Red
recombineering. These findings demonstrate the
guide-directed cleavage of pAgo on the host genome
is mutagenic and can lead to different outcomes ac-
cording to the function of the host DNA repair ma-
chinery. We anticipate this novel DNA-guided inter-
ference to be useful in broader genetic manipulation.
Our study also provides an
in vivo
assay to charac-
terize or engineer pAgo DNA cleavage activity.
INTRODUCTION
Prokaryotic argonaute proteins (pAgos) constitute a di-
verse protein family (
1
,
2
). Unlike their eukaryotic coun-
terparts, which use small RNA guides to interfere with
RNA targets in regulation and defense (
3
,
4
), many pA-
gos were reported to cleave DNA targets using small guide
DNAs (gDNAs)
in vitro
(
5–12
).
In vivo
, several pAgos
were shown to protect bacteria from foreign DNAs (
13–
17
), but the defense mechanism, especially whether double-
stranded DNA breaks (DSBs) are involved, remained elu-
sive until recently. In-depth insights into the mechanism of
pAgo-mediated defense were gained by analyzing CbAgo,
a pAgo nuclease from a mesophilic bacterium
Clostridium
butyricum
,in
Escherichia coli
as its expression host (
18
).
In that study, a DNA interference pathway was revealed in
CbAgo-mediated protection against invading DNAs. First,
CbAgo generates and binds gDNAs from plasmids or other
multicopy genetic elements. Next, gDNA-bound CbAgo in-
troduces DSBs at the homologous sites, including chromo-
somal regions, and causes DNA degradation in collabora-
tion with
E. coli
exonuclease RecBCD. Invader DNAs such
as plasmids and phages can thus be targeted and eliminated
efficiently through this mechanism.
It has been postulated that pAgo may have potential ap-
plications in genome manipulation ever since the discovery
of its DNA nuclease activity, which could represent a novel
DNA-guided genome editing tool that overcomes some of
the limitations of CRISPR-based methods such as unin-
tended secondary structures in guide RNA and difficulties
in guide RNA delivery (
5
,
19
). However, to the best of our
knowledge, mutations induced by guide-directed cleavage
of pAgos in the host genome have never been firmly estab-
lished. The observation that CbAgo can be directed to gen-
erate DSBs in chromosomes by plasmid-encoded guide se-
quences (GSs) motivated us to leverage such a mechanism
to manipulate
E. coli
genomes. Here we demonstrate that
guide-directed CbAgo cleavage can directly induce chromo-
some recombination between direct repeat sequences, or as-
sist Lambda-Red recombineering in
E. coli
as a counter-
selection. The recombination system described here can
also serve as an efficient
in vivo
assay to report or engineer
*
To whom correspondence should be addressed. Email: smayo@caltech.edu
Correspondence may also be addressed to Kaihang Wang. Email: kaihangwang@caltech.edu
Correspondence may also be addressed to Shan Huang. Tel: +1 650 725 9250; Fax: +1 650 724 1164; Email: gibbson783@gmail.com
Present address: Shan Huang, Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, St
anford Uni-
versity School of Medicine, Stanford, CA 94305, USA.
C
The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:
//
creativecommons.org
/
licenses
/
by
/
4.0
/
), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acids Research, 2023, Vol. 51, No. 8
4079
pAgo DNA nuclease activity as we find the recombination
rates from different pAgos correlate well with their reported
in vitro
cleavage activity. These findings demonstrate the po-
tential of establishing a DNA-directed genome editing sys-
tem using pAgo.
MATERIALS AND METHODS
Culture conditions
E. coli
, cultured in Luria-Bertani (LB) medium and agar,
was incubated at 37
◦
Cor30
◦
C. When appropriate, an-
tibiotics were added to the medium at the following final
concentrations: ampicillin, 100
g
/
ml; chloramphenicol, 20
g
/
ml; kanamycin, 35
g
/
ml. Bacterial cell growth was
monitored periodically by measuring the optical density of
culture aliquots at 600 nm.
Strains and plasmids
E. coli
strains used in this study are listed in Supplemen-
tary Table S1. Plasmids used in this study are listed in
Supplementary Tables S2. Oligonucleotides used in this
study are listed in Supplementary Tables S3. Procedures
for the construction of strains and plasmids are described
in Supplementary information. CbAgo-encoded plasmids
are pBR322-derived and have copy numbers
∼
15–20. Sup-
plementing 100
g
/
ml ampicillin in the growth media is
sufficient to maintain the plasmids, regardless of potential
CbAgo-mediated degradation.
Determination of recombination frequency
Cells were transformed with appropriate plasmids and
plated on LB plates supplemented with ampicillin. The next
day, 5 ml of LB medium supplemented with ampicillin was
inoculated with single colony and aerated at 37
◦
C until
OD
600
=
0.3–0.4. The temperature was then adjusted to
18
◦
C and after 30 min protein expression was induced by
adding anhydrotetracycline to 200 ng
/
ml for 16 h. Cultures
were then cooled down on ice for 10 min, washed with ice-
cold PBS (pH 7.2), resuspended in 5 ml of LB medium
supplemented with ampicillin, and recovered at 37
◦
Cfor
5 h. Serial dilutions of cells were plated on the LB plates
supplemented with appropriate antibiotics to determine
cfu.
Fluctuation analysis
Cells were transformed with appropriate plasmids and
plated on LB plates supplemented with ampicillin. The next
day, 1 ml of LB medium supplemented with ampicillin and
200 ng
/
ml anhydrotetracycline was inoculated with single
colony and aerated at 37
◦
C for 12 h before making serial
dilutions of cultures and plating on the LB plates supple-
mented with appropriate antibiotics to determine cfu.
For
plasmid-free
strain
3
×
ChikanS,
3
×
ChikanS
pal246
sbcCD and 3
×
ChikanS
pal246,
1 ml of LB medium was inoculated with single colony and
aerated at 37
◦
C for 12 h before making serial dilutions of
cultures and plating on the LB plates without antibiotic or
supplemented with kanamycin to determine cfu.
The Ma-Sandri-Sarkar Maximum Likelihood Estimator
(MSS-MLE) Method or the Lea-Coulson Method of the
Median in the Fluctuation AnaLysis CalculatOR (FAL-
COR) (
20
) were used to calculate recombination rates and
95% confidence intervals. The online FALCOR tool is avail-
able at
https://lianglab.brocku.ca/FALCOR/
.
Flow cytometry analysis
Strain SMR6669 cells were transformed with appropriate
plasmids and plated on LB plates supplemented with ampi-
cillin. The next day, 1 ml of LB medium supplemented with
ampicillin and 200 ng
/
ml anhydrotetracycline was inocu-
lated with single colony and aerated at 37
◦
C for 12 h. Cul-
tures were then washed with ice-cold PBS (pH 7.2), diluted
1:500 into ice-cold PBS (pH 7.2), passed through 40
m cell
strainers, added 1
g
/
ml propidium iodide to determine cell
viability, and analyzed on a Beckman Coulter Cytoflex S
Flow Cytometer. For each experiment, 10
5
cells per culture
and three cultures per strain were analyzed.
Flow cytometry data were analyzed using FlowJo soft-
ware version 10.8.1. To comparatively quantify green cells,
a green ‘gate’ was set arbitrarily as the window in which
∼
0.9% of the control strain, SMR6669
/
pEmpty fall, ac-
cording to the spontaneous SOS induction level (
21
).
Lambda-red recombineering
The kanamycin-resistance cassette was amplified from ge-
nomic DNA of SIJ488
lacZ via colony PCR with primers
Lambda.Red.F
/
Lambda.Red.R. Resulting PCR product
was gel-purified as dsDNA donor. To calculate mutation ef-
ficiency for Lambda-Red recombineering (referred to as the
standard recombineering procedure), 5 ml of LB medium
was inoculated with single colony of strain SIJ488
recA
and aerated at 37
◦
C until OD
600
=
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
l 10% glycerol. 2
l mixture of
∼
300 ng dsDNA was
added to the cells, which were then subject to electropora-
tion and allowed to recover in 1 ml LB for 2 h at 37
◦
C. Serial
dilutions of cells were plated on the LB plates with no an-
tibiotic or supplemented with kanamycin to determine cfu.
To calculate mutation efficiency for CbAgo-assisted
Lambda-Red recombineering, cells of strain SIJ488
recA
were transformed with appropriate plasmids and plated
on LB plates supplemented with ampicillin. The next day,
5 ml of LB medium supplemented with ampicillin was
inoculated with single colony and aerated at 37
◦
C until
OD
600
=
0.3–0.4. The Lambda-Red genes were then in-
duced 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
l 10% glycerol.
2
l mixture of
∼
300 ng dsDNA was added to the cells,
which were then subject to electroporation and allowed to
recover in 1 ml LB supplemented with ampicillin for 2 h at
37
◦
C. The recovered cells were diluted into 5 ml LB sup-
plemented with ampicillin and 0.2% glucose and continued
growing for 2 h at 37
◦
C. The temperature was then adjusted
to 18
◦
C and after 30 min protein expression was induced by
4080
Nucleic Acids Research, 2023, Vol. 51, No. 8
adding anhydrotetracycline to 200 ng
/
ml for 16 h. Upon
induction, cultures were cooled down on ice for 10 min,
washed with ice-cold PBS (pH 7.2), resuspended in 5 ml
of LB medium supplemented with ampicillin, and recov-
ered at 37
◦
C for 5 h. Serial dilutions of cells were plated on
the LB plates supplemented with appropriate antibiotics to
determine cfu.
Statistical analyses
GraphPad Prism 9 was used to evaluate statistical signifi-
cance. Student’s
t
-test (two-tailed) was used for the statisti-
cal analysis of experiments.
P
values
<
0.05 were considered
significant.
RESULTS
Creation of the recombination system
Previous observation that guide-directed CbAgo cleavage
at
E. coli
chromosomes efficiently triggers RecBCD activ-
ity (
18
) inspired us to hypothesize that RecBCD-dependent
chromosome recombination should be triggered by guide-
directed CbAgo cleavage as well. It has been shown that
DSBs introduced by SbcCD cleavage at a 246-bp chromo-
somal palindrome (pal246) stimulate RecBCD-dependent
recombination between two downstream direct repeat se-
quences (Supplementary Figure S1) (
22
). To determine
whether similar recombination can be induced by guide-
directed CbAgo cleavage (Figure
1
A, Supplementary Fig-
ure S2), we integrated a recombination cassette at cynX lo-
cus on
E. coli
strain DL1777, which is
∼
6kbawayfromthe
target lacZ locus (Figure
1
B). This recombination cassette
contains an EM7 promoter and a kanamycin resistance
gene whose function is abolished by the insertion of a stop
codon array, which is flanked by two 270-bp direct repeat
sequences. A recombination event between the two direct
repeat sequences removes the insertion, restores the gene
function, and confers kanamycin resistance to the host. It
has been demonstrated that the presence of a Chi site, an 8-
base 5
-GCTGGTGG-3
motif recognized by RecBCD (
23–
26
), near the direct repeat sequences stimulates recombina-
tion (
22
). Therefore, we incorporated varying numbers of
Chi sites into the genome with their 5
ends oriented towards
the recombination cassette (Figure
1
B).
To target the lacZ locus, we created a targeting plasmid
pTet
CbAgo
/
GS encoding a CbAgo expression cassette un-
der the control of a tetracycline-inducible promoter (pTet),
and a 1000-bp GS homologous to lacZ gene (Figure
1
C).
Importantly, GS is the only sequence on the plasmid (ex-
cept for an 80-bp rrnB T1 terminator sequence) that is ho-
mologous to the genome, assuring only the lacZ locus will
be effectively targeted. For controls, plasmids with no GS,
no CbAgo gene, or neither, were created. To determine the
dependence of CbAgo cleavage activity, we created plas-
mids encoding a CbAgo mutant (dCbAgo: CbAgo D541A-
D611A) that contains mutations of two catalytic residues
in its active site which were previously shown to abolish its
endonuclease activity
in vitro
(
6
,
7
) and DSB generation ac-
tivity
in vivo
(
18
).
We then combined the obtained plasmids and strains,
induced CbAgo expression, and recovered the induced
cells to measure recombination frequencies, which were
calculated as the fraction of ampicillin-resistant cells that
became resistant to kanamycin (kanamycin-resistant and
ampicillin-resistant colony-forming units (cfu)
/
ampicillin-
resistant cfu), because only the recombinants have restored
functional kanamycin resistance gene. When there are three
or six Chi sites adjacent to the recombination cassette (cor-
responding strain 3
×
ChikanS and 6
×
ChikanS), recombi-
nation frequencies by CbAgo
/
GS are significantly higher
than the rest control groups (Supplementary Figure S3A),
suggesting a recombination pathway that is mediated by
guide-directed CbAgo cleavage.
Interestingly, we observed remarkable recombination fre-
quencies in dCbAgo
/
GS groups in some conditions (Sup-
plementary Figure S3A). When there is no Chi site adja-
cent to the recombination cassette (corresponding strain
nonChikanS), the cell bearing dCbAgo
/
GS had a recom-
bination frequency being
∼
10-fold higher than the one
bearing CbAgo
/
GS. These results suggest there is a re-
combination pathway that is mediated by the non-cleavage
function of CbAgo and is outperformed by the cleavage-
dependent pathway in the presence of the CbAgo active
site. Although the exact mechanism remains unknown,
this dCbAgo-mediated recombination pathway should be
independent of DSB and RecBCD because dCbAgo
/
GS
was previously shown not able to generate DSB or trigger
RecBCD activity
in vivo
(
18
).
We also examined the effects of GS length on the recom-
bination frequency (Supplementary Figure S3B) and found
that in the range of 50–500 bp, recombination frequency
increases as GS length increases. We decided to use 1000
bp as the GS length and 3
×
ChikanS as the model strain
to perform fluctuation analysis (
20
) to estimate recombina-
tion rates (Figure
1
D). Fluctuation analysis, in general, pro-
vides an accurate estimate of mutation rate by calculating
mutation per generation, while recombination frequency re-
flects average mutant numbers among the population that
may vary greatly because mutants that arise earlier during
growth will expand more than those that arise later. The
cell bearing CbAgo
/
GS had a recombination rate that is
5-fold higher than the ones with dCbAgo, with and with-
out GS, 15-fold higher than the one with CbAgo-only, and
30-fold higher over the rest control groups. The actual con-
tribution of dCbAgo-mediated recombination to the total
recombination events in 3
×
ChikanS
CbAgo
/
GS should be
much smaller than one-fifth because it should be largely
outperformed by the cleavage-dependent pathway as pre-
vious observation suggests. Together, these findings reveal
novel chromosome recombination that is induced by guide-
directed CbAgo cleavage. Its Chi site dependence implies
DSB formation and RecBCD processing during recombi-
nation.
Validation of the recombination system
To demonstrate the reliability of our recombination system,
we tested four additional pAgos in strain 3
×
ChikanS, in-
cluding CaAgo, CdAgo, CpAgo and IbAgo (refer to Sup-
plementary Table S4 for the summary of pAgos used in this
study). In the presence of GS, the rates of recombination
induced by different pAgos correlate with the rank order of
Nucleic Acids Research, 2023, Vol. 51, No. 8
4081
B
lacZ
n×Chi
kan DR
Stop Codons
kan C-terminus
Recombination
D
C
CbAgo
GS
pTet_CbAgo/GS
A
(ii)
(iii)
Chi sites
Direct repeats
(v)
B
C
D
Target site
gDNA-loaded
CbAgo
Recombination rate
10
-4
10
-5
10
-6
GS
CbAgo
dCbAgo
+
-
Plasmid
GS
CbAgo
(i)
Chromosome
kan DR
(iv)
A
lacZ
n×Chi
kan DR
kan C-terminus
B
C
D
A
A
A
A
A
-
-
--
-
--
-
--
++
+
++
+
Figure 1.
Guide-directed CbAgo cleavage stimulates chromosome recombination. (
A
) Proposed mechanism for guide-directed CbAgo cleavage and
RecBCD-dependent recombination. (i) CbAgo acquires gDNA from plasmid-encoded GS. See Supplementary Figure S2 for possible mechanisms of
gDNA biogenesis. (ii) Guide-directed CbAgo cleavage of lagging strand during chromosome replication generates DSB and (iii) triggers RecBCD bindi
ng
and processing of chromosome DNA. (iv) Recognition of Chi sites by RecBCD attenuates its DNA degradation activity and triggers Holliday junction
formation with the help of RecA. (v) DNA replication and subsequent resolution of the Holliday junction result in recombination between sister chro-
mosomes and the deletion of the direct repeat sequence. (
B
) Structure of the recombination cassette. See materials and methods for DNA sequences. kan,
kanamycin resistance gene. DR, direct repeat. (
C
) Structure of the CbAgo targeting plasmid. (
D
) Recombination rates of strain 3
×
ChikanS in different
genetic contexts determined by fluctuation analysis from eight independent cultures. Error bars represent 95% confidence intervals.
their reported
in vitro
ssDNA cleavage activity (Figure
2
A,
see (
27
). We also tested an engineered DSB in our system
by integrating a pal246 into the lacZ locus on the genome
of strain 3
×
ChikanS and its
sbcCD
knockout mutant
and measuring their recombination rates (Figure
2
B, Sup-
plementary Figure S4). The sbcCD
+
, lacZ::pal246 strain
yielded a
∼
100-fold increase in recombination rate com-
pared to the sbcCD
+
,lacZ
+
strain and
sbcCD
, lacZ::pal246
strain. The bigger fold change stimulated by SbcCD
/
pal246
over CbAgo
/
GS is consistent with the previous observa-
tion that SbcCD
/
pal246 is more efficient in DSB genera-
tion than CbAgo
/
GS
in vivo
(
18
). These findings indicate
a strong correlation between pAgo DNA cleavage activity,
DSB generation efficiency, and recombination rate in our
system.
Recombination depends on DSB generation and RecBCD but
not RecA
To gain more insight into our recombination system, we first
sought to provide solid evidence that suggests CbAgo can
be directed to attack
E. coli
chromosomes and cause DNA
damage (Figure
1
A, steps i and ii). Since DNA damage in-
duces the cellular SOS response, we used an
E. coli
strain
4082
Nucleic Acids Research, 2023, Vol. 51, No. 8
A
10
-3
10
-4
10
-5
10
-6
B
pal246
SbcCD
Recombination rate
Recombination rate
CbAgo/GS CpAgo/GS
CaAgo/GS CdAgo/GS IbAgo/GS
6×10
-5
4×10
-5
2×10
-5
0
10
-4
8×10
-5
+
-
+
-
+
+
Figure 2.
Recombination rate by pAgo
/
GS correlates with pAgo
in vitro
DNA cleavage activity and
in vivo
DSB generation efficiency. (
A
)Re-
combination rates using different pAgos in the presence of GS in strain
3
×
ChikanS. The y-axis is on a linear scale to better visualize the differ-
ences among pAgo variants. See (
27
) for comparisons of in vitro DNA
cleavage activity among different pAgos. (
B
) Recombination rates us-
ing sbcCD
+
/
sbcCD, lacZ
+
/
lacZ::pal246 strains. Strains used here were
3
×
ChikanS, 3
×
ChikanS
pal246
sbcCD and 3
×
ChikanS
pal246. See
(
18
) for the comparison of
in vivo
DSB generation efficiency between
SbcCD
/
pal246 and CbAgo
/
GS. See Supplementary Figure S4 for the ge-
netic structure of engineered DSB. Recombination rates were determined
by fluctuation analysis from eight independent cultures. Error bars repre-
sent 95% confidence intervals.
carrying a chromosomally located gfp gene controlled by
an SOS-inducible
sulA
promoter (
21
) and performed flow
cytometry to quantify the single-cell fluorescence level (Fig-
ure
3
A). We observed a 6-fold increase of fluorescence in
cells expressing CbAgo and a 15-fold increase in cells con-
taining CbAgo
/
GS, while the cells containing dCbAgo
/
GS
exhibited no difference in cellular fluorescence level com-
pared to the cells bearing empty plasmids (Figure
3
B).
The fluorescence increase in cells expressing CbAgo with-
out GS can be explained by previous observations that
CbAgo actively degrades plasmids (
18
) and plasmids degra-
dation triggers SOS-response (
28
). Alternatively, CbAgos
that are loaded with chromosome-derived gDNAs can at-
tack chromosomes and trigger chromosome stress. Impor-
tantly, the significant increase of fluorescence in cells ex-
pressing CbAgo in the presence of GS confirms that CbAgo
can be guided to attack chromosomes, while dCbAgo
/
GS
cannot.
Then we sought to determine the involvement of
E. coli
endogenous DNA repair machinery RecA and
RecBCD in the recombination by creating and test-
ing 3
×
ChikanS
recA and 3
×
ChikanS
recBCD mutant
strains. Since the viabilities of knockout strains varied a
great deal after induction (Supplementary Figure S5), we
determined the fluctuation analysis is no longer suitable
and decided to directly analyze recombination frequen-
cies. For the 3
×
ChikanS
recBCD strain, there was no
difference in the recombination frequency of the CbAgo-
expressing cells in the presence or absence of GS. This is
a significant change from the result using the recBCD
+
strain (Figure
3
C), indicating the recombination induced
by CbAgo
/
GS depends on RecBCD (Figure
1
A, step iii).
Since RecBCD works closely with DSB (
29
), this obser-
vation also suggests DSB generation in CbAgo
/
GS in-
duced recombination. An interesting discovery was that the
3
×
ChikanS
recBCD strain bearing dCbAgo
/
GS showed
∼
1000-fold decreased ampicillin-resistant cfu and only
∼
20-fold decreased kanamycin-resistant and ampicillin-
resistant cfu compared to its RecBCD
+
counterpart (Sup-
plementary Figure S5). These changes resulted in increased
recombination frequency (Figure
3
C), suggesting dCbAgo-
mediated recombination is RecBCD-independent. More-
over, this growth inhibition was reduced by
∼
60-fold in the
presence of the CbAgo active site, suggesting it is outper-
formed or inhibited when the CbAgo active site is present.
The CbAgo
/
GS induced DSBs can further explain the
extremely low viability of 3
×
ChikanS
recA strain bear-
ing CbAgo
/
GS (Supplementary Figure S5): without the
protection of RecA, continuously introduced DSBs trig-
ger extensive DNA degradation by RecBCD, causing an
enormous loss of chromosomal DNA and subsequent cell
death (
30–32
). On the other hand, these cells had a high
recombination frequency close to 0.1 (Figure
3
C), indicat-
ing RecA is not essential in the recombination induced by
guide-directed CbAgo cleavage even though it actively re-
pairs DSBs generated during the process. These findings
motivated us to change the final step of our model (Figure
1
A, step v) to be independent of RecA. We hypothesize that
this step––the actual recombination step between the two
direct repeat sequences––may involve a mechanism simi-
lar to the RecA-independent, direct repeat-mediated DNA
deletion during replication arrest (
33
,
34
).
CbAgo cleavage assists recombineering
The observation that the cfu of 3
×
ChikanS
recA strain
was reduced by three orders of magnitude when its genome
is targeted by CbAgo (Supplementary Figure S5) is very in-
triguing, as it supports a strategy to leverage CbAgo
/
GS
targeting as a counter-selection to facilitate recombineer-
ing (Figure
4
A). For comparison, a self-targeting CRISPR-
Cas9 system was reported to reduce cfu by three orders
of magnitude in
E. coli
(
35
). Co-expressing the CRISPR-
Cas9 system to eliminate unedited cells, Lambda-Red re-
combineering achieved an increase of efficiency by
∼
10
4
fold and a 65% overall mutation rate. We sought to com-
bine the CbAgo targeting system with Lambda-Red recom-
bineering by introducing CbAgo expression plasmids into
strain SIJ488
recA, which is RecA-deficient and has ara-
binose inducible Lambda-Red recombineering genes inte-
grated into its genome. We first performed the standard
recombineering procedure with a dsDNA donor encod-
ing kanamycin resistance cassette to replace the genomic
Nucleic Acids Research, 2023, Vol. 51, No. 8
4083
C
A
GFP intensity
Empty
dCbAgo/GS
CbAgo
CbAgo/GS
B
10
-1
kan
R
, amp
R
cfu/amp
R
cfu
GS
10
-2
10
-3
10
-4
10
-5
10
-6
-
CbAgo
dCbAgo
-
-
+
-
-
-
--
-
--
+
+
+
++
+
Strain
3×ChikanS
****
****
****
n.s.
**
10
-1
10
1
10
3
10
5
80
60
40
20
0
100
Relative cell count
0
5
10
15
20
Empty
dCbAgo/GS
CbAgo
CbAgo/GS
Percentage GFP-positive
-
-
-
+
-
-
-
--
-
--
+
+
+
++
+
-
-
-
+
-
-
-
--
-
--
+
+
+
++
+
****
n.s.
*
**
**
3×ChikanS_∆recA
3×ChikanS_∆recBCD
Figure 3.
Recombination by CbAgo
/
GS depends on DSB generation and RecBCD processing but is independent of RecA. (
A
) CbAgo
/
GS induces cellular
SOS response. Flow cytometry histograms normalized to the mode of the population, combining data from three independent cultures. Horizontal bar
represents the GFP
+
gate. (
B
) Quantification of GFP-positive cells within the GFP
+
gate. Error bars, mean
±
s.d. from three independent cultures. (
C
)
Recombination frequencies in different genetic contexts. Error bars, mean
±
s.d. from eight independent cultures.
P
values were calculated by two-tailed
unpaired Student’s
t
-test; n.s.
P
>
0.05, *
P
<
0.05, **
P
<
0.01, ***
P
<
0.001, ****
P
<
0.0001.
lacZ gene. The recombineering efficiency was 1.2
×
10
−
4
,
calculated from the fraction of cells that became kanamycin
resistant. Then we performed recombineering in CbAgo-
plasmids contained cells, induced CbAgo expression, and
recovered the cells to characterize the proposed counter-
selection effect. The cell transformed with pTet
CbAgo
/
GS
had a mutation efficiency of 2.3
×
10
−
2
, representing a
∼
100-fold increase in efficiency from standard recombi-
neering (Figure
4
B). Other control groups did not yield im-
provement, therefore the increased proportion of the edited
cell population depends on guide-directed CbAgo cleavage.
DISCUSSION
Our study here demonstrates the combination of CbAgo
and plasmid-encoded GS can induce mutations in the
E.
coli
chromosome, via guide-directed CbAgo cleavage of tar-
get DNA, activation of DNA repair mechanism, and sub-
sequent chromosome recombination. This strategy may ap-
ply to other organisms if chromosomal DSBs can be intro-
duced and necessary cellular repair machinery can be trig-
gered. Besides, we also demonstrate the potential of pAgo
targeting to assist recombineering in RecA-deficient strains
as another genome editing strategy. This method may ex-
tend to RecA-active strains, if RecA activity can be effi-
ciently inhibited by, for example, expressing RecA inhibitor
(
36
). A recent study also reported NgAgo-assisted recom-
bineering (NgAgo, pAgo from
Natronobacterium gregoryi
),
but the fold change was smaller than 2 and the enhance-
ment of editing was not dependent on NgAgo endonuclease
activity (
37
).
The mechanism of guide-directed recombination by
dCbAgo in our system remains unknown, although this
pathway appears to be independent of RecBCD and DSB.
Since dCbAgo can load gDNAs from plasmids
in vivo
(
18
) (Supplementary Figure S2), there is a possibility that
dCbAgo may play a role in target recognition and follow-
ing recruitment of
E. coli
nuclease or recombinase. This
speculation is supported by findings in other pAgo research
(
14
,
38
,
39
) and the fact that many pAgo genes have been
found associated with a variety of genes including nuclease
and helicase (
1
,
13
).