Letter

pubs.acs.org/synthbio

Programmable DNA-Guided Artificial Restriction Enzymes

Behnam Enghiad† and Huimin Zhao*,†,‡
†
Department of Chemical and Biomolecular Engineering, ‡Carl R. Woese Institute for Genomic Biology, Department of Chemistry,
Department of Biochemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
*
S Supporting Information

ABSTRACT: Restriction enzymes are essential tools for

recombinant DNA technology that have revolutionized
modern biological research. However, they have limited
sequence specificity and availability. Here we report a
Pyrococcus f uriosus Argonaute (PfAgo) based platform for
generating artificial restriction enzymes (AREs) capable of
recognizing and cleaving DNA sequences at virtually any
arbitrary site and generating defined sticky ends of varying
length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by
reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and
molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts,
and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality,
versatility, and high efficiency for this new technology.
KEYWORDS: restriction enzymes, DNA cloning, DNA profiling, recombinant DNA technology

R estriction enzymes are bacterial proteins that recognize

specific DNA sequences and cut DNA at or near the
recognition site. They were discovered in bacteria in the late
cleases to cleave a target RNA molecule that is complementary
to their bound RNA guide at a specific location.10 In
prokaryotes, most currently characterized Argonaute proteins
1960s1 and have become the cornerstone of recombinant DNA are suggested to have a role in host defense against invasive
technology since the early 1970s.2 So far more than 3600 genetic elements.11 Unlike eukaryotic Argonautes, some
restriction enzymes with >250 distinct specificities have been prokaryotic Argonautes have the ability to use small DNA
characterized,3 in which >250 are now commercially available molecules instead of RNA as guides to cleave either a ssRNA or
for routine use in molecular biology. Most of them (i.e., type II ssDNA target.11c
restriction enzymes) only recognize short DNA sequences (4− In this work, we report the development of a new platform to
8 base pairs), which significantly limits their application in generate highly active AREs with virtually any sequence
recombinant DNA technology. specificity and defined sticky ends of varying length based on
To overcome this limitation, protein engineering was used to the prokaryotic Argonaute of the archaeon Pyrococcus f uriosus
alter the sequence specificity of naturally occurring restriction (PfAgo).11b We used this platform to generate over 18 AREs
enzymes, but met with limited success.4 Alternatively, artificial for DNA fingerprinting and molecular cloning of PCR-
restriction enzymes (AREs) have been generated by fusing a amplified or genomic DNAs.

■
DNA binding domain to a nuclease domain (e.g., zinc finger
nucleases (ZFNs)5 and transcription activator-like effector RESULTS AND DISCUSSION
nucleases (TALENs)6) or using the clustered regularly
interspaced short palindromic repeat (CRISPR)-associated PfAgo protein is a DNA-guided nuclease (771 amino acids)
enzyme 9 (Cas9) in vitro.7 Such AREs had higher specificity that targets cognate DNA and is most active at a temperature
than naturally occurring restriction enzymes. However, they did range from 87 to 99.9 °C.11b It utilizes small 5′-phosphorylated
not generate defined sticky or blunt ends, a hallmark of DNA guides to cleave single stranded DNA targets, and does
restriction enzymes, and showed rather poor activity due to not utilize RNA as guide or target. We sought to use this
their single or extremely low turnover numbers. Moreover, it is prokaryotic DNA-programmable nuclease system to generate a
difficult to obtain them in sufficient amount and purity. All new class of AREs capable of recognizing and cleaving DNA
these limitations significantly constrain their applications in sequences at virtually any arbitrary site and generating defined
vitro. sticky ends of varying length. Our strategy is to separate two
Argonaute proteins are a class of proteins that can bind short strands of DNA (circular or linear) by incubating the DNA
nucleotides and are conserved through all domains of life.8 In samples at high temperatures. After the two strands of DNA are
eukaryotes, Argonaute proteins are key players in RNA
silencing machinery.9 Eukaryotic Argonautes are able to bind Received: October 31, 2016
small RNA molecules and can function as guided endonu- Published: February 6, 2017

© 2017 American Chemical Society 752 DOI: 10.1021/acssynbio.6b00324

ACS Synth. Biol. 2017, 6, 752−757
ACS Synthetic Biology Letter

Figure 1. Overview of the PfAgo/ARE platform. Step 1: By incubating the reaction mixture at high temperatures (87−98 °C), the dsDNA target is
completely or partially denatured. PfAgo protein binds the 5′-phosphorylated ssDNA guides. Step 2: ssDNA guides bring PfAgo to their
corresponding targets. Upon binding, PfAgo is able to cleave each target at specific position. Step 3: After each strand cleavage, PfAgo leaves its target
DNA. Step 4: By decreasing the temperature, the two DNA strands reanneal and dsDNA cleavage is complete.

Figure 2. PCR cloning experiments. (A) An example of proof of concept PCR cloning using PfAgo/AREs. (B) Restriction digestion check for
correct cloning of PCR products digested with PfAgo(XbaI), PfAgo(NdeI), PfAgo(PfoI) AREs. Assembled plasmids were digested by corresponding
native restriction enzymes. (C) General map of pUC19 plasmid with positions of pUC19-g7, random target cloning sites and EcoRI digestion site
indicated. (D) Digestion analysis of negatively supercoiled pUC19 plasmid with PfAgo using EcoRI guides alone (lane 2) or EcoRI guides with
addition of pUC19-g7 (lane 3). pUC19 plasmid in the absence of guides and PfAgo was run as control (lane 1). (E) PfAgo-based digestion analysis
for correct cloning of PCR products at a random target. DNA guides used for PfAgo cleavage are shown with a black triangle. M: 1 kb DNA ladder.

partially or completely separated, PfAgo will use two different and used 16 nt guides for all the experiments. Like its other
phosphorylated DNA guides, targeting two strands of DNA at prokaryotic counterpart, TtAgo,11c PfAgo is believed to cleave
the desired locations, and cleave the strands in separate events. its ssDNA target between 10 and 11 nt position of its ssDNA
Once each strand of DNA is cleaved at the desired location, the guide (Figure S1A). To investigate specific cleavage by PfAgo,
two strands can reanneal to generate desired cleaved dsDNA we cleaved linearized pUC19 plasmid (linearized by NdeI)
(Figure 1). using two DNA guides that are expected to generate HindIII
PfAgo is able to use short phosphorylated DNA guides (>15 sticky ends. After cleavage by PfAgo using HindIII guides, the
nt) to cleave the ssDNA target. In our system, we codon- cleavage product was purified and cleavage ends were filled
optimized the PfAgo gene for high-level expression in E. coli using Klenow fragment polymerase. The resulting fragment was
753 DOI: 10.1021/acssynbio.6b00324
ACS Synth. Biol. 2017, 6, 752−757
ACS Synthetic Biology Letter

Figure 3. DNA fingerprinting experiments. (A) pCM2 DNA fingerprinting experiments. Linearized pCM2 (with Bsu36I) was digested with
combinations of PfAgo/AREs. The same reactions were run using respective restriction enzymes instead of PfAgo to serve as positive control (shown
by C). An overall map of linear pCM2 is also shown. (B) Lane 1: Linearized pCM2 was digested using PfAgo with AvrII, BamHI, XbaI guides. Lane
2: One of the XbaI guides was excluded from the reaction. Lane 3: One of the XbaI and one of the BamHI guides were excluded from the reaction.
(C) pUC19 fingerprinting experiment. Linearized pUC19 (with BsaI) was digested with PfAgo at three different random targets to create the correct
pattern (lane 1). M1:1 kb DNA ladder. M2:100 bp DNA ladder.

ligated to itself by blunt-end ligation and 40 assembled plasmids as our targets. For the first experiment, pUC19 plasmid was
were sent for DNA sequencing for cleavage site analysis. Out of linearized using either XbaI, EcoRI, or HindIII and then
the 40 plasmids, 39 were correctly assembled, and 38 of the dephosphorylated by shrimp alkaline phosphatase (rSAP). Each
plasmids showed cleavage at the expected location (Figure of the aforementioned genes was PCR amplified using primers
S1B). This high cleavage specificity of PfAgo makes generation to add 16 bp sequence of DNA corresponding to XbaI, EcoRI,
of defined sticky ends possible. or HindIII sites of pUC19 to each end and the amplified PCR
To determine PfAgo activity in cleaving dsDNA target, linear product was digested with PfAgo using two DNA guides to
pUC19 plasmid (linearized with XmnI) was cleaved using generate sticky ends of the corresponding restriction enzyme
different concentrations of PfAgo protein to generate EcoRI on each end. The digested fragment was then ligated into the
sticky ends. PfAgo cleaved 1 μg of linear pUC19 plasmid in less pUC19 backbone (Figure 2A) and the assembled plasmids
than 10 min at 95 °C when more than 3.75 pmol of the protein were analyzed by restriction digestion using the same restriction
was used (Figure S2A). In addition, > 100 nmol protein enzyme. More than 80% of the assembled plasmids showed the
(sufficient for >25 000 reactions) was readily purified from 1 L correct digestion pattern (Figure 2B and Figure S3). Such
of E. coli culture and PfAgo is known to be a multiple-turnover efficiency was comparable to native restriction enzymes for the
enzyme.11b Therefore, PfAgo is a highly active and readily same experiment (data not shown).
available protein for practical applications. To investigate It is known that XbaI, EcoRI, and HindIII all produce 5′
whether PfAgo is able to cleave methylated DNA target, linear sticky ends with 4 nt. To investigate whether sticky ends of
pUC19 plasmid (linearized with BsaI) was mixed with PfAgo other lengths can be generated by the PfAgo/AREs, the pUC19
and a set of DNA guides targeting an MboI restriction site on plasmid was digested with either NdeI or PfoI and then
the plasmid. MboI recognizes GATC as its target sequence but dephosphorylated with rSAP. NdeI generates 5′ sticky ends
is not able to cleave DNA when the target is methylated.3 with 2 nt while PfoI generates 5′ sticky ends with 5 nt. The xylE
PfAgo was able to cleave the methylated DNA target (Figure and Cas9 genes were PCR amplified with primers containing
S2B), demonstrating that PfAgo based AREs (PfAgo/AREs additional sequences corresponding to NdeI or PfoI sites on
thereafter) are not sensitive to DNA methylation. pUC19. The PCR products were then digested with PfAgo
To investigate whether PfAgo/AREs can be used for routine using NdeI or PfoI DNA guides. The digested PCR products
cloning of gene products amplified by polymerase chain were ligated into the digested and dephosphorylated vector
reaction (PCR), we selected xylE from Pseudomonas putida backbone and the assembled plasmids were analyzed by
(∼1 kb in size)12 and the Cas9 gene from Streptococcus pyogenes restriction digestion with appropriate restriction enzymes. In
(∼4 kb in size)13 as well as the commonly used plasmid pUC19 overall, more than 80% of the plasmids showed the correct
754 DOI: 10.1021/acssynbio.6b00324
ACS Synth. Biol. 2017, 6, 752−757
ACS Synthetic Biology Letter

digestion pattern, indicating that PfAgo/AREs can be used to same approach was used for the third and fourth experiments
generate arbitrary sticky ends (Figure 2B). by addition of XbaI and EcoRI guides to the reaction mixture
Next, we explored whether plasmid vector and PCR products until all 8 guides were present in the PfAgo reaction. PfAgo was
can both be digested with PfAgo using the same set of guides. able to generate the same pattern for the DNA target as
pUC19 plasmid was first incubated with PfAgo using EcoRI restriction enzymes using 8 guides in the same reaction. The
guides. PfAgo was not able to cleave the negatively supercoiled experiments were repeated multiple times with the same results
pUC19 plasmid using EcoRI guides. This result suggests that (Figure 3A).
even at high temperatures (90−100 °C) in PfAgo reaction One of the advantages of using PfAgo as a designer
buffer, the pUC19 plasmid strands could not be separated from restriction enzyme is the fact that generation of a double
each other at the EcoRI site to make the strands available for strand break with PfAgo requires the presence of both guides
PfAgo cleavage. To overcome this problem, another DNA targeting two different strands of DNA at positions close to
guide (pUC19-g7) was added to the reaction mixture. pUC19- each other. If one of the guides is not present in the reaction
g7 targets a region on the plasmid that has ∼29% GC content mixture, PfAgo would not be able to cleave the dsDNA and
(including a 20 bp flanking sequence on each side) (Figure would only create a nick on the target. This feature gives this
2C). Due to the fact that strand separation is more likely to
PfAgo/ARE platform great specificity because, for example,
occur at low GC content regions, PfAgo would be able to
generation of 3′ sticky ends of 4 nt requires two guides
generate a nick on the supercoiled plasmid at these sites. Once
targeting a 22 bp DNA sequence. To investigate whether this is
the nick is generated, the two strands would be partially or
completely separated and PfAgo would be able to cleave the actually the case, a set of DNA guides corresponding to AvrII,
nicked plasmid at any desired location. As expected, pUC19 BamHI, and XbaI sites on pCM2 was used for PfAgo cleavage.
could be cleaved by PfAgo at the EcoRI site using the additional In one experiment, one of the XbaI guides was excluded from
guide (Figure 2D). the reaction. In another experiment, one of the XbaI guides and
Using the aforementioned strategy, both pUC19 and the one of the BamHI guides were excluded from the reaction
PCR products were digested with PfAgo with a combination of mixture. The DNA patterns for each of these experiments
DNA guides (4 in total) corresponding to different sets of indeed confirmed that for DNA cleavage at one site, both
restriction enzyme cut sites including EcoRI and HindIII, guides must be present at the same time (Figure 3B).
BamHI and PfoI, EcoRI and NdeI, and XbaI and NdeI. The Linearized pUC19 (with BsaI) was also used for the
assembled plasmids were analyzed by restriction digestion using fingerprinting experiments. The pUC19 target was digested
the appropriate restriction enzyme set. In overall, more than with PfAgo using 6 guides in the same reaction in which each
60% of the colonies showed the correct color (i.e., white) in a set of guides were directed to generate sticky ends of varying
blue/white screening and more than 80% of the checked sizes. The digestion product showed the correct pattern based
plasmids showed the correct assembly (Figure S4). These on the known pUC19 sequence (Figure 3C), demonstrating
results indicate that PfAgo can be used as a single enzyme to the multiplexing ability of this PfAgo/ARE platform.
target a plasmid and PCR products at desired locations and can Finally, we tested whether PfAgo/AREs could be used for
replace the use of multiple restriction enzymes. direct cloning of genomic DNA targets because traditional
To demonstrate the versatility of this PfAgo/ARE platform, restriction enzyme-based cloning strategies are often limited by
both pUC19 and PCR products were digested at positions that the availability of unique restriction sites in the target DNA. To
no known restriction enzyme is able to cleave using two sets of this end, a random region from Streptomyces coelicolor A3(2)
DNA guides. Because no restriction enzyme is known to target genomic DNA with ∼2 kb in size was selected as a target. For
these sites, the assembled plasmids were analyzed by PfAgo/ this genomic DNA target, two sets of DNA guides were
ARE digestion using the same set of guides used for cloning designed. Each set of guides was expected to produce 5′ sticky
(Figure 2E). In overall, more than 70% of the colonies showed ends with the size of 4 nt on each end of the target. For receiver
the correct color (i.e., white) in a blue/white screening and generation, pUC19 plasmid was PCR amplified with primers
more than 80% of the checked plasmids showed the correct containing additional DNA sequences corresponding to each
digestion pattern. Taken together, these results demonstrate end of the genomic DNA target. Both the S. coelicolor genomic
that PfAgo/AREs can not only target the DNA regions that
DNA and the receiver were digested with PfAgo using the two
commercially available restriction enzymes can cleave, but also
sets of guides, and the purified DNA fragments were ligated
offer striking flexibility: DNA fragments can virtually be cleaved
together. The resulting plasmids were analyzed by restriction
at any desired location and assembled together using
appropriate sticky ends. digestion and DNA sequencing and 80% of them were correct
To explore whether PfAgo/AREs can be used for DNA (Figure S5).
fingerprinting, we selected a linear dsDNA with 10995 bp, i.e., In conclusion, we have established a PfAgo based platform
plasmid pCRISPomyces 2 (a.k.a. pCM2)14 linearized with for generating AREs with virtually any sequence specificity and
Bsu36I, as a target. pCM2 has unique restriction sites for the defined sticky ends of varying length and demonstrated their
restriction enzymes AvrII, BamHI, XbaI, and NdeI. Eight applications in DNA fingerprinting and DNA cloning. Due to
different phosphorylated DNA guides corresponding to each of the unprecedented simplicity, programmability, generality,
the restriction sites were designed and a set of digestion versatility, and multiplexing ability (a single protein plus
experiments with PfAgo were performed (Figure 3A). For each DNA guides for targeting any one or more sites) as well as
experiment, the same DNA target was digested with the accessibility (easy high-level purification of the PfAgo protein
restriction enzymes instead of PfAgo/AREs to serve as positive and inexpensive synthesis of short DNA guides), we expect
control. In the first experiment, the linearized target was cleaved PfAgo/AREs will become a powerful and indispensable tool in
using PfAgo and AvrII guides. In the second experiment, all restriction enzyme or broadly speaking, nuclease enabled
BamHI guides were also added to the reaction mixture. The basic and applied biological research and medicine.
755 DOI: 10.1021/acssynbio.6b00324
ACS Synth. Biol. 2017, 6, 752−757
ACS Synthetic Biology Letter

■ METHODS
PfAgo Expression and Purification. A strep(II)-tagged
all digestion reactions, the molar ratio of overall DNA guides to
PfAgo was kept higher than 10:1. The reaction mixture was
incubated at 95 °C for 10 min followed by slow cooling. The
(N-terminal) codon-optimized version of PfAgo gene was
digested PCR products were purified using Qiaquick PCR
ordered from GeneScript (Piscataway, NJ). The codon-
purification kit (QIAGEN). Purified products were ligated into
optimized gene was cloned into pET28a plasmid to yield the
the corresponding digested backbones using T4 DNA ligase
expression plasmid pHZ-PfAgo. The expression plasmid was
(New England Biolabs). For cas9 ligation, the ligation mixture
transformed into Escherichia coli KRX (Promega) according to
was incubated overnight at 16 °C. For xylE ligation, the ligation
manufacturer’s protocol. The strain was cultivated overnight at
mixture was incubated at room temperature for 15 min. After
37 °C in LB medium supplemented with 0.4% (w/v) glucose
ligation, all the reaction mixtures were transformed into NEB5α
and 50 μg/mL kanamycin. Following overnight incubation, the
competent E. coli (New England Biolabs) according to
culture was centrifuged at 3220g for 5 min and the supernatant
manufacturer’s protocol.
was removed. Cell pellets were resuspended in Terrific Broth pUC19 Digestion with PfAgo/AREs. For all pUC19 plasmid
containing 50 μg/mL kanamycin and incubated at 37 °C until digestions, pUC19-g7 was added to the reaction mixture. For
the OD600 of 1.2−1.5 was reached. The culture was cold all cloning experiments with the vector digested by PfAgo/
shocked by incubation in ice bath for 15 min and protein AREs, ∼1 μg of pUC19 plasmid was digested in a 50 μL
expression was induced by addition of isopropyl β-D-1- reaction containing PfAgo with the final concentration of ∼0.2
thiogalactopyranoside (IPTG) and L-rhamnose to final μM. The ratio of pUC19-g7 to all other guides was kept at 1:2
concentrations of 1 mM and 0.1% (w/v), respectively. and the molar ratio of overall DNA guides to PfAgo was kept
Expression was continued by incubation at 30 °C for 20 h. higher than 10:1. The reaction mixtures were incubated at 95
Purification was performed using previously mentioned °C for 15 min followed by slow cooling. Digested vectors were
protocol11b with minor modification in the sonication step purified by agarose gel extraction and purification using
(20 30 s pulses at 30% power with 30 s pause between pulses). Zymoclean Gel DNA recovery Kit.
The purified protein was stored in storage buffer (20 mM Tris- Analysis of Correct Plasmid Assembly Using PfAgo/AREs.
HCl, pH 8.0, 300 mM NaCl, 0.5 mM MnCl2, 15% (v/v) For digestion analysis of the plasmids assembled in random
glycerol) and the aliquots were stored at −80 °C. The purified target cloning, between 700 ng to 1 μg of the assembled
protein sample was run on SDS-PAGE and the resolved bands plasmids were mixed in the reaction buffer supplemented with
are shown in Figure S6. 5% (v/v) DMSO and PfAgo with the final concentration of 0.2
Restriction Digestion Using PfAgo/AREs and Native μM. The ratio of pUC19-g7 to all other guides was kept at 1:2
Restriction Enzymes. For all PfAgo digestions (unless noted and the molar ratio of overall DNA guides to PfAgo was kept
otherwise), the same reaction buffer (20 mM HEPES pH 7.50, higher than 10:1. The reaction mixtures were incubated at 87
250 mM NaCl, 0.5 mM MnCl2) was used. PfAgo, ssDNA °C for 30 min followed by incubation at 95 °C for 10 min. The
guides, and target dsDNA were mixed together in the reaction samples were cooled down slowly until 10 °C was reached.
buffer and incubated at high temperatures (87 to 98 °C). Afterward, the reaction mixtures were mixed with purple
Following high temperature incubation, the samples were loading dye (New England Biolabs) and were run on agarose
cooled down by slowly lowering the temperature with the rate gel electrophoresis.
of 0.1 °C/s until 10 °C was reached. For all digestion reactions, DNA Fingerprinting Using PfAgo/AREs. pCM2 plasmid
the molar ratio of overall DNA guides to PfAgo was kept higher was first digested with Bsu36I and purified using Qiaquick PCR
than 10:1 and all guides were mixed in equimolar ratios with purification kit (QIAGEN). For all fingerprinting experiments,
respect to each other (unless noted otherwise). A titration around 1 μg of the linearized target was mixed in 20 μL
experiment for optimum guide to protein ratio was performed reaction buffer with PfAgo concentrations ranging from 0.1 to
on linear pUC19 target (linearized by XmnI) using HindIII 0.2 μM. The molar ratio of overall DNA guides to PfAgo was
DNA guides and the results are shown in Figure S7. For all kept higher than 10:1. Reaction mixtures were incubated at 95
native restriction enzymes, the reactions were performed °C for 10 min followed by slow cooling. Upon completion of
according to manufacturer’s protocols. HindIII-HF, EcoRI-HF, the digestion, the samples were mixed with purple loading dye
XbaI, NdeI, AvrII, BamHI-HF, Bsu36I, BsaI-HF, MboI, and (New England Biolabs) and were run on agarose gel
XmnI restriction enzymes were ordered from New England electrophoresis. For PfAgo/AREs digestion with 8 guides at
Biolabs (Ipswich, MA). For PCR cloning experiments, the same time, the reaction was incubated at 95 °C for 15 min
FastDigest PfoI and BamHI enzymes were purchased from instead of 10 min.
Thermo Fisher Scientific (Waltham, MA). rSAP, DNA For pUC19 fingerprinting, the plasmid was first digested with
polymerase I, Klenow fragment, 1 kb and 100 bp DNA ladders BsaI and purified using Qiaquick PCR purification kit
were purchased from New England Biolabs. (QIAGEN). Around 500 ng of the digested pUC19 was
PCR Cloning Using PfAgo/AREs. PCR Products Diges- mixed in 20 μL reaction buffer with PfAgo concentration of 0.2
tion with PfAgo. The xylE and cas9 genes were PCR amplified μM and the sample was incubated at 95 °C for 20 min followed
using the appropriate primers mentioned in Supplementary by slow cooling. The molar ratio of overall DNA guides to
Table S1. PCR reactions were performed by Q5 Hot Start PfAgo was kept at 12:1.
DNA Polymerase (New England Biolabs) according to Genomic DNA Cloning Using PfAgo AREs. S. coelicolor
manufacturer’s protocol. The PCR products were purified by A3(2) genomic DNA was purified using Wizard genomic DNA
agarose gel extraction using Zymoclean Gel DNA recovery Kit purification kit (Promega). Around 20 μg of the genomic DNA
(Zymo Research). Purified PCR products were digested with was mixed in 400 μL reaction buffer containing PfAgo at the
PfAgo AREs. For either xylE or cas9, between 500 ng to 1 μg of concentration of 0.2 μM. For the 2 kb genomic DNA target,
the purified PCR product was digested in a 50 μL reaction ectoine(2)-g1, ectoine(2)-g2, ectoine(2)-g3, and ectoine(2)-g4
containing PfAgo with the final concentration of ∼0.2 μM. For were used as ssDNA guides. The overall guide to PfAgo ratio
756 DOI: 10.1021/acssynbio.6b00324
ACS Synth. Biol. 2017, 6, 752−757
ACS Synthetic Biology Letter

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405−12.
(11) (a) Olovnikov, I., Chan, K., Sachidanandam, R., Newman, D. K.,
ASSOCIATED CONTENT and Aravin, A. A. (2013) Bacterial argonaute samples the tran-
*
S Supporting Information scriptome to identify foreign DNA. Mol. Cell 51 (5), 594−605.
The Supporting Information is available free of charge on the (b) Swarts, D. C., Hegge, J. W., Hinojo, I., Shiimori, M., Ellis, M. A.,
Dumrongkulraksa, J., Terns, R. M., Terns, M. P., and van der Oost, J.
ACS Publications website at DOI: 10.1021/acssynbio.6b00324.
(2015) Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided
Additional methods, figures, and tables (PDF) nuclease that targets cognate DNA. Nucleic Acids Res. 43 (10), 5120−9.

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(c) Swarts, D. C., Jore, M. M., Westra, E. R., Zhu, Y. F., Janssen, J. H.,
AUTHOR INFORMATION Snijders, A. P., Wang, Y. L., Patel, D. J., Berenguer, J., Brouns, S. J. J.,
and van der Oost, J. (2014) DNA-guided DNA interference by a
Corresponding Author prokaryotic Argonaute. Nature 507 (7491), 258−261.
*Phone: (217) 333-2631. Fax: (217) 333-5052. E-mail: (12) Ingram, C., Brawner, M., Youngman, P., and Westpheling, J.
zhao5@illinois.edu. (1989) xylE functions as an efficient reporter gene in Streptomyces Spp
ORCID - use for the study of galp1, a catabolite-controlled promoter. J.
Huimin Zhao: 0000-0002-9069-6739 Bacteriol. 171 (12), 6617−6624.
(13) Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A.,
Author Contributions and Charpentier, E. (2012) A programmable dual-RNA-guided DNA
B.E. and H.Z. designed the experiments and wrote the endonuclease in adaptive bacterial immunity. Science 337 (6096), 816−
manuscript. B.E. performed the experiments. 821.
Notes (14) Cobb, R. E., Wang, Y., and Zhao, H. (2015) High-efficiency
The authors declare no competing financial interest. multiplex genome editing of Streptomyces species using an engineered

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CRISPR/Cas system. ACS Synth. Biol. 4 (6), 723−8.
ACKNOWLEDGMENTS
This work was supported by the Steven L. Miller Chair
Endowment (H.Z.). B.E. acknowledges fellowship support from
3M Corporation. B.E and H.Z. have filed a patent application
related to this work.

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757 DOI: 10.1021/acssynbio.6b00324

ACS Synth. Biol. 2017, 6, 752−757