A novel tool for microbial genome editing using the ... - ACS Publications

Subscriber access provided by Queen Mary, University of London

Article

A novel tool for microbial genome editing using the restriction-modification system Hua Bai, Aihua Deng, Shuwen Liu, Di Cui, Qidi Qiu, Laiyou Wang, Zhao Yang, Jie Wu, Xiuling Shang, Yun Zhang, and Tingyi Wen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00254 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1

A novel tool for microbial genome editing using the restriction-modification

2

system

3

Hua Bai†,‡, Aihua Deng†,*, Shuwen Liu†, Di Cui†,‡, Qidi Qiu†,‡, Laiyou Wang†,‡, Zhao

4

Yang†, Jie Wu†, Xiuling Shang† , Yun Zhang† and Tingyi Wen†,§,*

5 6

†

7

Microbiology, Chinese Academy of Sciences, Beijing 100101, China

8

‡

University of Chinese Academy of Sciences, Beijing 100049, China

9

§

Savaid medical school, University of Chinese Academy of Sciences, Beijing,

10

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of

100049, China

11 12

Graphical Table of Contents

13 14

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

15

ABSTRACT

16

Scarless genetic manipulation of genomes is an essential tool for biological research.

17

The restriction-modification (R-M) system is a defense system in bacteria that

18

protects against invading genomes based on its ability to distinguish foreign DNA

19

from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE)

20

technique for scarless genetic manipulation in different microorganisms. For bacteria

21

with Type IV REase, an RMGE technique using the inducible DNA methyltransferase

22

gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to

23

edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE

24

technique based on a restriction endonuclease (RMGE-mcrA) was established in

25

Bacillus subtilis. These techniques were successfully used for gene deletion and

26

replacement with nearly 100% counter-selection efficiencies, which were higher and

27

more stable compared to conventional methods. Furthermore, precise point mutation

28

without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a

29

single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA

30

technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414

31

with 100% counter-selection efficiency. The effectiveness of the RMGE technique in

32

E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this

33

technique for microbial genome manipulation.

34 35

KEYWORDS: scarless genome editing, counter-selection cassette,

36

restriction-modification (R-M) system, Escherichia coli, Bacillus subtilis,

37

Saccharomyces cerevisiae 2


Page 2 of 34

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38 39

INTRODUCTION

40

Understanding the function of genes1 or developing rational design for biological

41

engineering2 in bacteria heavily depends on the technology to edit the DNA sequence.

42

To modify the DNA sequence, numerous genetic tools have been developed to

43

manipulate bacterial chromosomes3-5. At early stages, a selectable marker was

44

employed to introduce a chromosomal mutation, but the number of available markers

45

limited multigene modifications. Although site-specific recombinases (FLP or Cre)

46

have been used to excise marker, a scar, such as a FRT or loxP site, is left in the

47

chromosome6. To overcome this problem, scarless genome editing techniques are

48

particularly valuable to introduce defined mutations, especially single nucleotide

49

mutations7.

50

Among the recently developed methods for scarless genome editing without any

51

limiting sites, the counter-selectable system is a common technique in various bacteria,

52

such as Escherichia coli7, Bacillus subtilis8, 9, and Corynebacterium glutamicum10, etc.

53

However, most of the existing counter-selectable systems are insufficient to introduce

54

precise mutations in bacterial genomes. For example, the counter-selectable system

55

mediated by rpsL11, pheS12, thyA13, tolC14, or lacY15 requires a special modification of

56

the chromosome to prepare the prerequisite mutants, which is time-consuming and

57

could limit its application in strains without clear genetic backgrounds. Although the

58

sacB-based counter-selectable system can be directly used in the target strain, the

59

counter-selection efficiency of the method is low7. In addition, the meganuclease 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60

I-SceI that recognizes an 18-bp specific sequence

61

counter-selection marker for scarless genetic manipulation through introducing the

62

recognition site16, 17 . Recently, a toxin-antitoxin (TA) system, a type of defense

63

system in bacteria and archaea, has been used as an effective and stable genome

64

manipulation techniques7, 8. However, they are inefficient to meet the demands of

65

bacteria with different genetic backgrounds. Therefore, an efficient, universal, and

66

scarless genome editing technique is still urgently needed for genetic manipulation of

67

bacteria with different genetic backgrounds.

has been used as a

68

The restriction-modification (R-M) system is a group of defense systems that

69

consists of DNA methyltransferases (MTases) and restriction endonucleases (REases).

70

These systems exist both in bacteria and archaea, and more than 96% of

71

genome-sequenced bacteria possess R-M systems18. R-M systems have been

72

classified into four types based on their subunit composition, cleavage sites, sequence

73

specificity, and cofactors. Type I, II, and III REases cleave DNA without methylation

74

at specific sequences, and Type IV REases cleave DNA with an exogenous

75

methylation pattern19. R-M systems protect bacteria from invading exogenous DNA20,

76

and therefore, this defensive machinery can be used to cleave the bacterial genome in

77

a similar way to the clustered, regularly interspaced, short palindromic repeats

78

(CRISPR) systems21. Unlike the CRISPR systems that can cleave genomic DNA at a

79

single site directed by a single-guide RNA (sgRNA), R-M systems are able to cleave

80

the genome at multiple sites22, 23. In general, exogenous REases can kill target cells by

81

cleaving their genomic DNA and can be potentially used as a counter-selectable 4


Page 4 of 34

Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


82

marker in a wide range of bacteria. Furthermore, for bacteria with Type IV REases,

83

exogenous MTases can also kill target cells due to their exogenous methylation of

84

genomic DNA, which is then cleaved by the Type IV REase of the host bacteria24.

85

Accordingly, both exogenous REases and MTases can be used as counter-selectable

86

markers in bacteria with Type IV REases. Therefore, the specific regulation

87

mechanisms of the R-M system play pivotal roles in cell growth and death and thus

88

might be powerful tools in genetic engineering for both fundamental research and

89

application purposes.

90

In this study, we developed an R-M system-mediated genome editing technique

91

(designated as RMGE) for bacteria with or without Type IV REases. Using the model

92

bacterium E. coli as an example of bacteria with Type IV REases (Table S1), a

93

RMGE system based on inducible Type II MTase BceSIIM (designated as REGE-

94

bceSIIM) was developed for genome editing in E. coli K-12 derivative W3110.

95

Furthermore, the RMGE system based on the inducible Type IV REase McrA25

96

(designated as REGE-mcrA) was constructed to manipulate the genome of the model

97

Gram-positive bacterium B. subtilis W168, which was chosen as an example of

98

bacteria without Type IV REases (Table S1). Additionally, RMGE-mcrA could also

99

be used to manipulate the genome of Saccharomyces cerevisiae DAY414. Our results

100

demonstrated that RMGE was convenient and efficient for genome editing in E. coli,

101

B. subtilis and S. cerevisiae, suggesting its great potential for application in a broad

102

range of microorganisms.

103 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

104

RESULTS

105

Design of R-M system-mediated genome editing (RMGE) for bacteria with or

106

without Type IV REases. According to the genetic background of the target bacteria,

107

a suitable R-M system was selected as a counter-selectable marker for RMGE. For

108

example, E. coli W3110 contains Type IV REases (Figure 1a; Table S1) that can

109

cleave DNA with exogenous methylation patterns. Thus, an exogenous REase or

110

MTases can be used as the counter-selectable marker in genome editing of these

111

bacteria. Our previous study showed that a Type II MTase BceSIIM from B. cereus

112

ATCC 10987 can be expressed in E. coli26. When the bceSIIM gene is expressed in

113

the E. coli, a m5C residue in the sequence GGWCC (W=A or T)26 of the genomic

114

DNA is introduced by the BceSIIM and then cleaved by Type IV REases McrBC,

115

which kills target cells 27. Therefore, the MTase BceSIIM was employed as a

116

counter-selection cassette to construct the RMGE genome editing tool in bacteria with

117

Type IV REases.

118

As an effective counter-selection cassette for genetic manipulation, MTase only

119

works in bacteria with Type IV REases, but not in bacteria without Type IV REases,

120

such as B. subtilis W168 (Figure 1b; Table S1). The genomic DNA of W168 is

121

methylated in its own pattern, so the exogenous REases that can cleave the genome

122

could be used as the counter-selection cassette. For example, when the REase McrA is

123

integrated into the genome and expressed in B. subtilis, the sequence YCGR (the

124

recognition sequence A(/G)CGC(/T) of McrA) 28 of the genome is cleaved into pieces

125

by the REase, which kills target cells. Therefore, various REases had to be screened 6


Page 6 of 34

Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


126

as a counter-selection cassette to construct the RMGE genome editing tool in bacteria

127

without Type IV REases.

128 129

Construction of an RMGE system based on the inducible MTase BceSIIM

130

(RMGE- bceSIIM). To assess the feasibility of the R-M system in genome editing of

131

E. coli, MTase BceSIIM was selected to construct the counter-selection cassette. The

132

temperature-sensitive plasmid pWYE3000, which is lost during high-temperature

133

cultivation (Figure S1), was firstly constructed as a recombinant vector by overlap

134

PCR. To further construct the counter-selection cassette based on MTase BceSIIM,

135

the bceSIIM gene, controlled by the arabinose-inducible promoter Para, was

136

PCR-amplified from WYE700 and ligated into pWYE3000 (Figure 2a). The

137

pWYE3001 plasmid was generated then transferred into E. coli W3110 to test the

138

regulation of cell growth and death by inducible bceSIIM. As shown in Figure 2b, no

139

growth was detected in the strain EC3004 (W3110/ pWYE3001) after the addition of

140

arabinose, suggesting that the inducible bceSIIM can effectively regulate the host

141

cell’s growth and death. Thus, MTase BceSIIM can be used for genetic manipulation

142

of E. coli, and the pWYE3001 plasmid was used as the universal genome editing

143

vector for various gene modifications.

144

According to the standard operation procedure (Figure 3a), recombinant plasmids

145

were constructed by inserting up- and down-stream homologous arms of the target

146

gene into the universal vector pWYE3001 and transferred into W3110. Transformants

147

were selected on LB medium with chloramphenicol (Cm) at 30 °C for 10 h. To obtain 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

148

an integrated strain through a single crossover event, positive transformants harboring

149

the recombinant plasmid were cultured in 5 mL of LB medium with Cm at 30 °C for

150

10 h. The liquid culture was then plated on LB plates with Cm after being diluted 10-3

151

times and incubated at 42 °C for 12 h. After PCR verification, the plasmid-integrated

152

strain was cultured in 5 mL of LB at 30 °C for 12 h. Then, the liquid culture was

153

diluted by 10-4−10-5 and grown on LB plates with the appropriate concentration of

154

arabinose at 30 °C for 12 h for counter selection. The plasmid-integrated strain

155

expressing exogenous MTase were killed by the Type IV REases. The colonies that

156

grew on the LB plate but failed to grow on the LB plate with Cm were verified by

157

PCR amplification to select positive mutant strains. DNA sequencing of the mutated

158

sites and the physiological changes of the resulting mutants were also examined for

159

further verification.

160 161

Scarless single gene deletion in E. coli. To validate the RMGE- bceSIIM system, a

162

diaminopimelate decarboxylase encoding gene, lysA, was chosen as the target to edit

163

the chromosome of E. coli W3110. The resulting mutant EC3007 (W3110∆lysA) was

164

obtained with 100% counter-selection efficiency, a similar efficiency to the traditional

165

SacB system (Table S2 and Figure S2a). Physiological changes of the mutant EC3007

166

were further identified in MM medium. In contrast to the normal growth of W3110,

167

the mutant EC3007 could not grow on MM plates, whereas it could grow on MM

168

plates supplemented with lysine (Figure 3b). The results indicated that the mutant

169

could not synthetize lysine due to deletion of the lysA gene. 8


Page 8 of 34

Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


170

Because the target sites might affect the counter-selection efficiency, another two

171

genes, lacZ and tolC, encoding beta-galactosidase and outer membrane protein,

172

respectively, were selected to further verify the RMGE- bceSIIM system. The

173

lacZ-deficient strain EC3010 (W3110∆lacZ) was generated with 100%

174

counter-selection efficiency, which was significantly higher compared to the 4.5 ± 4.0%

175

achieved using the SacB system (Table S2 and Figure S2b). The color of the mutant

176

EC3010 on LB plates with X-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside)

177

did not turn blue, further confirming the absence of beta-galactosidase activity

178

through deletion of the lacZ gene (Figure 3c). Furthermore, the tolC-deficient strain

179

EC3048 (W3110∆tolC) was obtained with 100% counter-selection efficiency, higher

180

than the73.7 ± 6.2% achieved by using the SacB system (Figure S2c). Because TolC

181

can confer the host cell with resistance to sodium dodecyl sulfate (SDS), the

182

sensitivity of the mutant EC3048 to SDS was identified on LB medium with 0.01%

183

SDS (Figure 3d).

184 185

Precise point mutation and gene replacement in E. coli. To evaluate the feasibility

186

for precise point mutations using the RMGE- bceSIIM system, a single base mutation

187

(A128C) was introduced into the rpsL gene encoding the small subunit ribosomal

188

protein S12, which confers streptomycin resistance. Following standard procedures,

189

the mutant EC3013 (W3110 rpsLA128C) was obtained with 100% counter-selection

190

efficiency (Table S2; Figure S3a). The resistance of the mutant to streptomycin was

191

tested, and the nucleotide sequence of rpsL was further confirmed by DNA 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

192 193

sequencing (Figure 4a). To assess the potential of RMGE- bceSIIM for gene replacement, the gfpmuat3a

194

gene controlled by the promoter PPL29 was used to replace the promoter of the frdA

195

gene encoding the fumarate reductase flavoprotein subunit. A recombinant plasmid,

196

pWYE3005, with homologous arms and a fragment of PPL-gfpmuat3a was obtained.

197

The mutant strain EC3016 (W3110 PfrdA::PPL-gfpmut3a) was obtained by introducing

198

the pWYE3005 into W3110 with 100% counter-selection efficiency, which was

199

higher than the 59.7 ± 5.1% obtained with the SacB system (Table S2; Figure S3b).

200

The GFP intensity was further detected by flow cytometry analysis, which confirmed

201

the expression of the gfpmut3a gene in the mutant EC3016 (Figure 4b).

202

To further assess the potential of RMGE- bceSIIM for metabolic engineering, the

203

cadA gene controlled by the promoter PPL was used to replace the region between the

204

ygcE and ygcF genes. The mutant strain EC3053 (W3110 ygcE-ygcF::PPL-cadA) was

205

generated with 98.7 ± 1.1% counter-selection efficiency, higher than the 84.6 ± 8.8%

206

efficiency obtained with the SacB system (Figure S3c). Because lysine can be

207

catalyzed by CadA and converted into cadaverine, accumulated cadaverine in EC3053

208

was measured to be 3.35 ± 0.14 g/L, which was significantly higher than that of

209

W3110 (Figure 4c and d).

210 211

Construction of a RMGE system based on the inducible REase McrA (RMGE-

212

mcrA). To develop a general method in bacteria without Type IV REase, REases were

213

used to construct a counter-selection cassette for B. subtilis W168. Type IV restriction 10


Page 10 of 34

Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


214

endonucleases from E. coli W3110, including McrBC, McrA, and Mrr, were selected

215

as candidates to evaluate their effectiveness25, 30. Three REase genes were separately

216

inserted into the plasmid pHCMC04 under control of the xylose-inducible promoter.

217

The lethal effects of the three REases on W168 were further detected on LB plates

218

with Cm and xylose after the expression plasmids were introduced into the target cells.

219

Figure 5a showed that the strain BS232 (W168/pWYE3008) did not grow on plates

220

with Cm and xylose, indicating that the REase McrA inhibited the growth of W168.

221

Thus, the inducible mcrA gene was selected as an effective counter-selection cassette

222

to establish the RMGE-mcrA system. A suicide plasmid, pWYE3010, which was

223

constructed by removing the replication origin reP from the plasmid pWYE3008

224

(Figure 5b), was used as a universal genome editing vector for genetic manipulation

225

in B. subtilis.

226 227

Genetic manipulation in B. subtilis using the RMGE- mcrA system. For genetic

228

manipulation in B. subtilis W168, the basic principle of scarless genome editing was

229

illustrated in Figure 6a. The plasmid-integrated strain was obtained by

230

electro-transformation of the recombinant plasmid into W168. Afterwards, the

231

plasmid-integrated strain was cultured in LB medium for 12 h and then on LB plates

232

with xylose at 30 °C for 12 h after first being diluted by a factor of 10-4-10-5 for

233

counter selection. Cells that contained the counter-selection cassette mcrA would be

234

killed, whereas colonies without mcrA were identified by PCR amplification and

235

DNA sequencing of the mutated sites. 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

236

To evaluate the efficiency of gene editing in B. subtilis using RMGE, the amyE

237

gene encoding α-amylase was chosen as a target for deletion. The recombinant

238

plasmid was constructed by inserting the up- and down-stream homologous arms of

239

the amyE gene into pWYE3010. The mutant BS235 (W168∆amyE) was obtained with

240

100% of counter-selection efficiency (Figure S4a). The mutant BS235 was further

241

verified by PCR amplification and DNA sequencing. No clear halo was formed in the

242

mutant BS235 by the starch plate assay compared to the original W168, further

243

confirming the lack of α-amylase (Figure 6b).

244

To assess the efficiency of RMGE in gene replacement, the gfpmuat3a gene was

245

chosen to replace the upp gene in W168. The mutant strain BS237 was generated with

246

100% counter-selection efficiency (Figure S4b). The GFP intensity was further

247

detected by flow cytometry analysis, which showed the expression of the gfpmut3a

248

gene in the mutant BS237 (Figure 6c).

249 250

Genome editing in Saccharomyces cerevisiae using RMGE-mcrA system. To test

251

whether RMGE can be applied in S. cerevisiae, McrA from E. coli W3110 as well as

252

BceSIIR and BceSIIIR from B. cereus ATCC 10987 were selected as candidates to

253

perform genome editing in S. cerevisiae. Three REase genes were separately cloned

254

into the plasmid pYES2/CT under the control of galactose-inducible promoter to test

255

the effect of REase gene expression on the cell growth. As shown in Figure 7a, the

256

strain SC201 (DAY414/pWYE3023) could not grow on the SG-Ura plate, indicating

257

that the expression of REase McrA resulted in the growth inhibition of DAY414. 12


Page 12 of 34

Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


258

Thus, the mcrA gene was selected as an effective counter-selection cassette to

259

construct the plasmid pWYE3026 in which the replication origin 2μ was removed

260

from the plasmid pWYE3023 (Figure 7b).

261

To evaluate the efficiency of RMGE-mcrA system in S. cerevisiae, CAN1 gene

262

encoding the plasma membrane arginine permease that imports the toxic compound

263

L-canavanine

264

constructed by inserting the up- and down-stream homologous arms of the CAN1 gene

265

into pWYE3026. The mutant SC205 (DAY414∆ CAN1) was obtained with 100% of

266

counter-selection efficiency (Figure S6). The mutant SC205 was further verified by

267

PCR amplification and DNA sequencing. The result showed that SC205 was able to

268

grow on synthetic complete medium containing L-canavanine due to the deletion of

269

the CAN1 gene (Figure 7c).

was chosen as a target for deletion. The recombinant plasmid was

270 271

Discussion

272

Here we firstly developed an R-M system-mediated genome editing (RMGE)

273

technique for scarless genetic manipulation in E. coli W3110, B. subtilis W168 and S.

274

cerevisiae DAY414. Different types of mutations, such as deletions, replacements,

275

and point mutations, were successfully achieved. The advantages of RMGE are as

276

follows: (i) it has a high and stable counter-selection efficiency compared with

277

traditional methods, such as counter-selection systems mediated by sacB, mazF9, and

278

relE8; (ii) it is versatile and can be widely used in bacteria and yeasts with or without

279

Type IV REases ; and (iii) it can edit genomes at any loci without the limitation of a 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

280

protospacer-adjacent motif (PAM), such as CRISPR. Therefore, the RMGE technique

281

holds promise as an efficient, versatile, and affordable strategy to perform

282

sophisticated genetic modifications in a broad range of bacteria and yeasts.

283

In this study, RMGE was efficient for deletion, replacement, and point mutation

284

of 6 target loci in E. coli with nearly 100% counter-selection efficiencies, which were

285

significantly increased compared to those of SacB (4.5-100%) and RelE (87-100%)

286

systems (Table S3). This method was also efficient in B. subtilis for gene deletion and

287

replacement with 100% counter-selection efficiencies, which were more stable than

288

that of TCCRAS and RelE systems (Table S4). However, mutation efficiencies of

289

RMGE were varied for all 9 target loci in both E. coli, B. subtilis and S. cerevisiae,

290

and were not comparable to existing methods, such as homologous

291

recombination-based counter-selection systems and nuclease-mediated CRISPR/Cas9

292

systems (Table S3, 4 and 5). The high and stable counter-selection efficiency

293

achieved by RMGE might be attributed to the strong ability of killing target cells

294

mediated by R-M systems. In general, the R-M systems can restrict the growth of

295

phage31 and cause post-segregational killing of host strains23. Thus, the R-M systems

296

can cleave DNA efficiently as shown in Figure 2b and 5a. Except for the strains

297

harboring the expression plasmids, this lethal effect was also detected in the strains

298

that had integrated a single copy of the inducible R-M system, which further

299

suggested that the R-M system can cleave DNA efficiently (Figure S5).

300

The RMGE technique is a versatile tool that can be used in a broad range of

301

bacteria. To our knowledge, the vast diversity and prevalence of R-M systems in the 14


Page 14 of 34

Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


302

prokaryotic kingdom18 are evidence of their success in preventing exogenous DNA

303

invasions23. The horizontal transfer32, 33 of many R-M systems indicates that the R-M

304

system has great potential for expression in heterologous species26. Based on this

305

characteristic of R-M systems, the bceSIIM and mcrA genes were successfully

306

heterologously expressed in E. coli W3110 (Figure 2b) and B. subtilis W168 (Figure

307

5a), respectively. Therefore, RMGE- bceSIIM and RMGE- mcrA systems can be

308

applied in E. coli W3110 and B. subtilis W168, respectively, and the RMGE

309

technique may be universally useful for genome manipulation in other bacterial

310

strains.

311

Genome editing mediated by R-M systems is also affordable. R-M systems used

312

for genome editing are based on the modification pattern of the target bacterial

313

genome. Different R-M systems should be chosen for genome editing in bacteria with

314

different genetic backgrounds. Information regarding R-M systems and the

315

methylation patterns of bacteria can be found in REBASE18 from genome sequencing

316

efforts, which makes it easy to construct a counter-selection cassette mediated by

317

R-M systems. Moreover, mutants achieved by RMGE only require one transformation

318

for any loci in the bacterial chromosomes.

319

In principle, the REGM technique can be established by using an exogenous

320

REase as the universal counter-selection cassette both in bacteria with and without

321

Type IV REases. In our preliminary study, BceSIIR34, a Type II REase that cleaves

322

the sequence GGWCC without methylation by BceSIIM, was used to construct the

323

universal counter-selection cassette. However, the expression plasmid harboring the 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

324

bceSIIR gene caused host cell death and was not generated due to its leaky expression

325

in E. coli. To solve this problem, the MTase bceSIIM was chosen as the

326

counter-selection marker for genome editing in E. coli. Additionally, the universal

327

counter-selection cassette could also be established by engineering a tightly regulated

328

inducible system.

329

RMGE can also be applied to genome editing of yeasts without R-M system, such

330

as S. cerevisiae. Due to the deficiency of Type IV REases in S. cerevisiae, the

331

exogenous MTases could not be used as a counter-selection marker. However, the

332

mouse-methylated DNA could be cleaved by McrBC27, indicating that REases had

333

potential to regulate the growth and death of the organisms without R-M system. As a

334

result, McrA was screened as the counter-selectable marker to successfully delete the

335

CAN1 gene in S. cerevisiae DAY414 (Figure 7a). Therefore, RMGE might be useful

336

for genome manipulation in other organisms without R-M system.

337

In summary, a novel strategy of RMGE was developed for scarless genome

338

editing in bacteria and yeasts with or without Type IV REase. Various mutations,

339

including deletions, replacements, and point mutations were achieved with nearly 100%

340

counter-selection efficiencies in E. coli, B. subtilis, and S. cerevisiae by the RMGE

341

technique using the MTase BceSIIM and REase McrA as the counter-selection

342

cassettes, respectively. Therefore, the RMGE approach is an efficient, versatile, and

343

affordable tool for genome editing in a broad range of microorganisms.

344 345

MATERIALS AND METHODS 16


Page 16 of 34

Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


346

Bacterial strains, plasmids, and primers. The bacterial strains and plasmids used in

347

this study are listed in Table S6. EC135, which lacks all R-M systems and orphan

348

MTases, was used to construct the plasmids. All of the primers (Table S7) used in this

349

study were synthesized by Invitrogen (Shanghai, China). DNA sequencing was

350

performed by Beijing Genomics Institute (BGI, Beijing, China).

351 352

Media and culture conditions. E. coli W3110 (F-λ- rph-1Inv(rrnD-rrnE))35 and B.

353

subtilis W168 (trp+)36 were cultured in Luria-Bertani (LB) medium. The

354

concentrations of the antibiotics used in this study were as follows: 100 µg/mL

355

ampicillin and 34 µg/mL Cm for E. coli, and 10 µg/mL Cm for B. subtilis. To induce

356

the promoters ParaB and PxylA, 0.2% arabinose and 1% xylose, respectively, were used.

357

Minimal medium (MM; 5 g/L glucose, 1 g/L Na3C6H5O7·2H2O, 2 g/L (NH4)2SO4, 0.2

358

g/L MgSO4·7H2O, 14 g/L K2HPO4·3H2O, 6 g/L KH2PO4, 100 µg/L biotin, 5 µmol/L

359

MnSO4, and 0.4 µg/mL thiamine, pH 7.1) was used to identify auxotrophic mutants.

360

Yeast was cultured in YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L

361

glucose). YPG medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L galactose)

362

was used for culturing the yeast in counter-selection process. Synthetic complete

363

medium lacking uracil (SC-Ura) was used to select yeast transformants. Synthetic

364

complete galactose agar lacking uracil plates (SG-Ura) were used for testing the

365

regulation of S. cerevisiae cell growth and death.

366 367

DNA manipulation. To construct the plasmid pWYE3000, two DNA fragments of 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

368

rep101- cmR and f1 origin were amplified by polymerase chain reaction (PCR) and

369

then fused together by overlap PCR to obtain the linear plasmid pWYE3000. The

370

linear plasmid was circularized by DNA ligase in vitro and transferred into EC135 by

371

the calcium chloride technique37. The DNA fragment containing the bceSIIM gene

372

and promoter ParaB was PCR-amplified from pWYE700 and inserted into pWYE3000

373

to generate the plasmid pWYE3001, which was used for genomic modification in E.

374

coli. To obtain the plasmid pWYE3010 for genomic modification in B. subtilis, a PCR

375

fragment of the mcrA gene was inserted into the pHCMC04 plasmid to yield

376

pWYE3008. Then, the replication initiation protein-encoding gene repA was deleted

377

from pWYE3008 by PCR to yield the pWYE3010 plasmid. The recombinant

378

plasmids were constructed by inserting the up- and down-stream homologous arms of

379

the target gene (or/and the inserted or mutated fragments) into the universal genome

380

editing vectors. Transformations of E. coli, B. subtilis, and S. cerevisiae were

381

separately performed as described by Kirill et al38, Zhaopeng Sun et al39 and Gietz et

382

al40.

383 384

Counter-selection efficiencies. After the recombinant plasmid had been integrated

385

into the target strain, positive transformants were subjected to counter-selection by

386

growing them in 5 mL of LB medium for 12 h. Then, the culture was plated on the LB

387

with an inducer after dilution by 105 fold. To calculate the counter-selection

388

efficiency, 156 colonies were randomly selected from the LB plate and subcultured on

389

LB plates with or without antibiotics. Counter-selection efficiency was defined as the 18


Page 18 of 34

Page 19 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


390

ratio of colonies that had excised the counter-selection cassettes to the total number of

391

colonies tested on the LB plate. The calculation formula was as follows:

392

Counter–selection efficiency = (Number of colonies growing on LB plate –

393

Number of colonies growing on LB plate with antibiotics)/ Number of colonies

394

growing on LB plate

395 396

Flow cytometry analysis. The gfpmut3a gene encoding an enhanced green

397

fluorescent protein (GFP) was used as a reporter gene. Flow cytometry was performed

398

on a BD FACSCalibur flow cytometer (Becton, Dickinson and Company, the United

399

States). Mutants integrating the gfpmut3a gene were cultured in LB media for 12 h,

400

resuspended to an OD600 of 0.4 using PBS buffer (137 mmol/L NaCl, 2.7 mmol/L

401

KCl, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4, pH 7.2), and placed on ice prior to

402

analysis. A total of 50,000 events were collected for each sample. E. coli W3110 and

403

B. subtilis W168 were used as controls to determine the background fluorescence.

404 405

Fermentation in shake flasks. The E. coli mutant EC3053 (W3110

406

ygcE-ygcF::PPL-cadA) was cultured in 500-mL shake flasks containing 50 mL of LB

407

media. When the OD600 reached 4.5, 5% (vol/vol) of the culture was seeded into a

408

500-mL baffled shake flask containing 50 mL of fermentation media (20 g/L glucose,

409

10 g/L lysine, 10 g/L (NH4)2SO4, 2 g/L KH2PO4, 2 g/L MgSO4, 15 g/L CaCO3, and 2

410

g/L yeast extract). Throughout the experiment, 2 mL aliquots of the culture were

411

collected to analyze cell growth by a spectrophotometer as well as the cadaverine 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

412

concentration by HPLC as described by Jie Wu et al8. The wild-type strain W3110

413

was used as the negative control. All experiments and measurements were performed

414

at least in triplicate.

415 416 417

AUTHOR INFORMATION

418

Corresponding Authors

419

*E-mail: [email protected]; [email protected]

420 421

Notes

422

The authors declare no financial conflicts of interest.

423 424

ACKNOWLEDGEMENTS

425

The authors would like to thank Tong Zhao (Institute of Microbiology, Chinese

426

Academy of Sciences) for assistance with the flow cytometry analysis. This work was

427

supported by the National Natural Science Foundation of China (31570083 and

428

31170103), National Hi-Tech Research and Development Program of China

429

(2014AA021203), Science and Technology Service Network Initiative

430

(KFJ-STS-QYZD-047 and KFJ-EW-STS-078), and the Key Deployment Projects of

431

Chinese Academy of Sciences (KGZD-EW-606).

432 433

ASSOCIATED CONTENT

434

Supporting Information 20


Page 20 of 34

Page 21 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


435

Supporting Information is available free of charge on the ACS Publications website.

436

Table S1. The MTases and REases of target strains in this study.

437

Table S2. Counter-selection efficiencies of the genome-scale engineering using the

438

RMGE system.

439

Table S3. Counter-selection efficiencies and mutation efficiencies of the RMGE

440

system in E. coli.

441


442

system in B. subtilis.

443


444

system in S. cerevisiae.

445

Table S6. Bacterial strains and plasmids used in this study

446

Table S7. Primers used in this study.

447

Figure S1. Temperature sensitive assay of the plasmid pWYE3000.

448

Figure S2. Counter-selection efficiencies for the gene-deficient strains in E. coli.

449

Figure S3. Counter-selection efficiencies for point mutation and gene replacement in

450

E. coli.

451

Figure S4. Counter-selection efficiencies for genome editing in B. subtilis by RMGE.

452

Figure S5. The lethal effect detected in the strains that had integrated a single copy of

453

the inducible R-M system in the chromosome.

454

Figure S6. Counter-selection efficiencies for deletion of the CAN1 gene in S.

455

cerevisiae by RMGE

456 457 458 459 460

REFERENCES 1

Haimovich, A. D., Muir, P., and Isaacs, F. J. (2015) Genomes by design, Nat Rev Genet 16, 501-516. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

2

Song, C. W., Lee, J., and Lee, S. Y. (2015) Genome engineering and gene expression control

3

Sharan, S. K., Thomason, L. C., Kuznetsov, S. G., and Court, D. L. (2009) Recombineering: a

for bacterial strain development, Biotechnol J 10, 56-68. homologous recombination-based method of genetic engineering, Nat Protoc 4, 206-223. 4

Yan, X., Yu, H. J., Hong, Q., and Li, S. P. (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis, Appl Environ Microbiol 74, 5556-5562.

5

Cheng, Z., Yi, P., Wang, X., Chai, Y., Feng, G., Yang, Y., Liang, X., Zhu, Z., Li, W., and Ou, G. (2013) Conditional targeted genome editing using somatically expressed TALENs in C. elegans, Nat Biotechnol 31, 934-937.

6

Kilby, N. J., Snaith, M. R., and Murray, J. A. (1993) Site-specific recombinases: tools for

7

Khetrapal, V., Mehershahi, K., Rafee, S., Chen, S., Lim, C. L., and Chen, S. L. (2015) A set of

genome engineering, Trends in genetics : TIG 9, 413-421. powerful negative selection systems for unmodified Enterobacteriaceae, Nucleic Acids Res 43, e83. 8

Wu, J., Deng, A., Sun, Q., Bai, H., Sun, Z., Shang, X., Zhang, Y., Liu, Q., Liang, Y., Liu, S., Che, Y., and Wen, T. (2017) Bacterial genome editing via a designed toxin-antitoxin cassette, ACS Synth Biol, 10.1021/acssynbio.1026b00287.

9

Jeong, D. E., Park, S. H., Pan, J. G., Kim, E. J., and Choi, S. K. (2015) Genome engineering using a synthetic gene circuit in Bacillus subtilis, Nucleic Acids Res 43, e42.

10

Ma, W., Wang, X., Mao, Y., Wang, Z., Chen, T., and Zhao, X. (2015) Development of a markerless gene replacement system in Corynebacterium glutamicum using upp as a counter-selection marker, Biotechnol Lett 37, 609-617.

11

Heermann, R., Zeppenfeld, T., and Jung, K. (2008) Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red(R)/ET(R) recombination, Microb Cell Fact 7, 14.

12

Miyazaki, K. (2015) Molecular engineering of a PheS counterselection marker for improved operating efficiency in Escherichia coli, Biotechniques 58, 86-88.

13

Wong, Q. N., Ng, V. C., Lin, M. C., Kung, H. F., Chan, D., and Huang, J. D. (2005) Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli, Nucleic Acids Res 33, e59.

14

DeVito, J. A. (2008) Recombineering with tolC as a selectable/counter-selectable marker: remodeling the rRNA operons of Escherichia coli, Nucleic Acids Res 36, e4.

15

Murphy, C. K., Stewart, E. J., and Beckwith, J. (1995) A double counter-selection system for the study of null alleles of essential genes in Escherichia coli, Gene 155, 1-7.

16

Janes, B. K., and Stibitz, S. (2006) Routine markerless gene replacement in Bacillus anthracis, Infect. Immun. 74, 1949-1953.

17

Yu, B. J., Kang, K. H., Lee, J. H., Sung, B. H., Kim, M. S., and Kim, S. C. (2008) Rapid and efficient construction of markerless deletions in the Escherichia coli genome, Nucleic Acids Res 36.

18

Roberts, R. J., Vincze, T., Posfai, J., and Macelis, D. (2015) REBASE--a database for DNA restriction and modification: enzymes, genes and genomes, Nucleic Acids Res 43, D298-299.

19

Roberts, R. J., Belfort, M., Bestor, T., Bhagwat, A. S., Bickle, T. A., Bitinaite, J., Blumenthal, R. M., Degtyarev, S. K., Dryden, D. T. F., Dybvig, K., Firman, K., Gromova, E. S., Gumport, R. I., Halford, S. E., Hattman, S., Heitman, J., Hornby, D. P., Janulaitis, A., Jeltsch, A., 22


Page 22 of 34

Page 23 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548

Josephsen, J., Kiss, A., Klaenhammer, T. R., Kobayashi, I., Kong, H. M., Kruger, D. H., Lacks, S., Marinus, M. G., Miyahara, M., Morgan, R. D., Murray, N. E., Nagaraja, V., Piekarowicz, A., Pingoud, A., Raleigh, E., Rao, D. N., Reich, N., Repin, V. E., Selker, E. U., Shaw, P. C., Stein, D. C., Stoddard, B. L., Szybalski, W., Trautner, T. A., Van Etten, J. L., Vitor, J. M. B., Wilson, G. G., and Xu, S. Y. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res 31, 1805-1812. 20

Labrie, S. J., Samson, J. E., and Moineau, S. (2010) Bacteriophage resistance mechanisms, Nat Rev Microbiol 8, 317-327.

21

Jiang, W., Bikard, D., Cox, D., Zhang, F., and Marraffini, L. A. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems, Nat Biotechnol 31, 233-239.

22

Naito, T., Kusano, K., and Kobayashi, I. (1995) Selfish behavior of restriction-modification systems, Science 267, 897-899.

23

Vasu, K., and Nagaraja, V. (2013) Diverse functions of restriction-modification systems in addition to cellular defense, Microbiol. Mol. Biol. Rev. 77, 53-72.

24

Fukuda, E., Kaminska, K. H., Bujnicki, J. M., and Kobayashi, I. (2008) Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases, Genome Biol 9, R163.

25

Raleigh, E. A., Murray, N. E., Revel, H., Blumenthal, R. M., Westaway, D., Reith, A. D., Rigby, P. W. J., Elhai, J., and Hanahan, D. (1988) McrA and McrB restriction phenotypes of some Escherichia coli strains and implications for gene cloning, Nucleic Acids Res 16, 1563-1575.

26

Zhang, G., Wang, W., Deng, A., Sun, Z., Zhang, Y., Liang, Y., Che, Y., and Wen, T. (2012) A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria, PLoS Genet 8, e1002987.

27

Raleigh, E. A. (1992) Organization and function of the mcrBC genes of Escherichia coli K-12, Mol. Microbiol. 6, 1079-1086.

28

Mulligan, E. A., Hatchwell, E., McCorkle, S. R., and Dunn, J. J. (2010) Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG, Nucleic Acids Res 38, 1997-2005.

29

Alper, H., Fischer, C., Nevoigt, E., and Stephanopoulos, G. (2005) Tuning genetic control through promoter engineering, Proc Natl Acad Sci U S A 102, 12678-12683.

30

Waiterees, P. A., Keating, C. J., Moran, L. S., Slatko, B. E., Hornstra, L. J., and Benner, J. S. (1991) Characterization and expression of the Escherichia coli mrr restriction system, J Bacteriol 173, 5207-5219.

31

Bogdanova, E., Zakharova, M., Streeter, S., Taylor, J., Heyduk, T., Kneale, G., and Severinov, K. (2009) Transcription regulation of restriction-modification system Esp1396I, Nucleic Acids Res 37, 3354-3366.

32

Lee, K. F., Shaw, P. C., Picone, S. J., Wilson, G. G., and Lunnen, K. D. (1998) Sequence comparison of the EcoHK31I and EaeI restriction-modification systems suggests an intergenic transfer of genetic material, Biol Chem 379, 437-441.

33

Jeltsch, A., and Pingoud, A. (1996) Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems, J Mol Evol 42, 91-96.

34

Xu, S. Y., Nugent, R. L., Kasamkattil, J., Fomenkov, A., Gupta, Y., Aggarwal, A., Wang, X., Li, Z.,

Zheng,

Y.,

and

Morgan,

R.

(2012)

Characterization

23


of

type

II

and

III


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567

restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579, J Bacteriol 194, 49-60. 35

Hayashi, K., Morooka, N., Yamamoto, Y., Fujita, K., Isono, K., Choi, S., Ohtsubo, E., Baba, T., Wanner, B. L., Mori, H., and Horiuchi, T. (2006) Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110, Mol. Syst. Biol. 2, 2006 0007.

36

Zeigler, D. R., Pragai, Z., Rodriguez, S., Chevreux, B., Muffler, A., Albert, T., Bai, R., Wyss, M., and Perkins, J. B. (2008) The origins of 168, W23, and other Bacillus subtilis legacy strains, J bacteriol 190, 6983-6995.

37

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 53-84.

38

Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci U S A 97, 6640-6645.

39

Sun, Z., Deng, A., Hu, T., Wu, J., Sun, Q., Bai, H., Zhang, G., and Wen, T. (2015) A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35, Appl Microbiol Biotechnol 99, 5151-5162.

40

Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method, In Guide to Yeast Genetics and Molecular and Cell Biology, Pt B (Guthrie, C., and Fink, G. R., Eds.), pp 87-96, Elsevier Academic Press Inc, San Diego.

568 569

24


Page 24 of 34

Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


570

Figures legends

571

Figure 1. Schematic illustration of RMGE for genetic manipulation. (a) A schematic

572

illustration of genome editing by RMGE in bacteria with Type IV REase. (b) A

573

schematic illustration of genome editing by RMGE in bacteria without Type IV

574

REase. R represents A or G, Y represents C or T and W represents A or T.

575 576

Figure 2. Construction and functional characterization of the plasmid pWYE3001. (a)

577

Construction of the plasmid pWYE3001. The plasmid pWYE3000 was constructed by

578

removing the sacB gene and a useless DNA fragment (marked in the gray) from the

579

plasmid pKOV. The fragment of the inducible bceSIIM gene was inserted into

580

pWYE3000 to generate the plasmid pWYE3001. (b) The regulation effect of the

581

bceSIIM gene on cell growth/death in E. coli. The strain EC3003 (W3110/pWYE3000)

582

was used as the control.

583 584

Figure 3. Genome editing in E. coli using RMGE- bceSIIM. (a) Schematic illustration

585

of the standard operating procedure for genetic manipulation in E. coli. UHA and

586

DHA represent the up- and down-stream homologous arms, respectively. The

587

asterisks indicate the deletion, replacement, or point mutation. (b) MM plate assay for

588

the deletion of the lysA gene. Due to lacking the lysA gene, the mutant EC3007 cannot

589

grow on the MM plate unless 0.5 g/L lysine has been added. (c) The deletion of the

590

lacZ gene was detected on LB plates with 0.1 g/L X-gal. (d) The deletion of the tolC

591

gene was detected by testing the sensitivity of the mutant EC3048 to SDS. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

592 593

Figure 4. Point mutation and gene replacement using RMGE- bceSIIM. (a)

594

Verification of the mutant strain EC3013 (W3110 rpsLA128C) by testing for

595

streptomycin resistance and DNA sequencing. (b) Flow cytometry analysis of the

596

expression of gfpmut3a in EC3016 (W3110frdA::PPL- gfpmut3a). (c) Detection of

597

cadaverine produced by EC3053 (W3110ygcE-ygcF::PPL-cadA) and W3110 by

598

HPLC. (d) Cell growth and cadaverine production of W3110 and EC3053.

599 600

Figure 5. Design and construction of the plasmid pWYE3010 in B. subtilis. (a)

601

Screening for an effective REase to inhibit cell growth in B. subtilis. The strain W168

602

containing pHCMC04 was used as a control. (b) Construction of the plasmid

603

pWYE3010. The plasmid pWYE3010 cannot replicate in B. subtilis because the rep

604

gene fragment was removed.

605 606

Figure 6. Genome editing in B. subtilis using RMGE- mcrA. (a) Schematic

607

illustration of the standard operating procedure for genetic manipulation in B. subtilis.

608

UHA and DHA represent the up- and down-stream homologous arms, respectively.

609

The asterisks indicate a deletion or replacement. (b) Detection of the amyE gene

610

deletion using starch plates. The strains were cultured on LB plates containing 1%

611

starch for 12 h, and then the plate was stained with iodine. W168 was used as a

612

control. (c) Flow cytometry analysis of the expression of GFP in BS237

613

(W168upp::P43-gfpmut3a). 26


Page 26 of 34

Page 27 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


614

Figure 7. Genome editing in S. cerevisiae using RMGE- mcrA. (a) Screening for an

615

effective REase to regulate cell growth and death in S. cerevisiae. The strain DAY414

616

containing pYES2.0/CT was used as a control. (b) Construction of the plasmid

617

pWYE3026. The plasmid pWYE3026 was generated by removing the 2µ gene

618

fragment from pWYE3023. (c) Detection of the CAN1 gene using synthetic complete

619

medium plates with 60 µg/mL L-canavanine. DAY414 was used as a control.

620

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Schematic illustration of RMGE for genetic manipulation. (a) A schematic illustration of genome editing by RMGE in bacteria with Type IV REase. (b) A schematic illustration of genome editing by RMGE in bacteria without Type IV REase. R represents A or G, Y represents C or T and W represents A or T. 107x64mm (300 x 300 DPI)


Page 28 of 34

Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Construction and functional characterization of the plasmid pWYE3001. (a) Construction of the plasmid pWYE3001. The plasmid pWYE3000 was constructed by removing the sacB gene and a useless DNA fragment (marked in the gray) from the plasmid pKOV. The fragment of the inducible bceSIIM gene was inserted into pWYE3000 to generate the plasmid pWYE3001. (b) The regulation effect of the bceSIIM gene on cell growth/death in E. coli. The strain EC3003 (W3110/pWYE3000) was used as the control. 240x325mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Genome editing in E. coli using RMGE- bceSIIM. (a) Schematic illustration of the standard operating procedure for genetic manipulation in E. coli. UHA and DHA represent the up- and down-stream homologous arms, respectively. The asterisks indicate the deletion, replacement, or point mutation. (b) MM plate assay for the deletion of the lysA gene. Due to lacking the lysA gene, the mutant EC3007 cannot grow on the MM plate unless 0.5 g/L lysine has been added. (c) The deletion of the lacZ gene was detected on LB plates with 0.1 g/L X-gal. (d) The deletion of the tolC gene was detected by testing the sensitivity of the mutant EC3048 to SDS. 139x109mm (300 x 300 DPI)


Page 30 of 34

Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Point mutation and gene replacement using RMGE- bceSIIM. (a) Verification of the mutant strain EC3013 (W3110 rpsLA128C) by testing for streptomycin resistance and DNA sequencing. (b) Flow cytometry analysis of the expression of gfpmut3a in EC3016 (W3110frdA::PPL- gfpmut3a). (c) Detection of cadaverine produced by EC3053 (W3110ygcE-ygcF::PPL-cadA) and W3110 by HPLC. (d) Cell growth and cadaverine production of W3110 and EC3053. 168x159mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Design and construction of the plasmid pWYE3010 in B. subtilis. (a) Screening for an effective REase to inhibit cell growth in B. subtilis. The strain W168 containing pHCMC04 was used as a control. (b) Construction of the plasmid pWYE3010. The plasmid pWYE3010 cannot replicate in B. subtilis because the rep gene fragment was removed. 141x112mm (300 x 300 DPI)


Page 32 of 34

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Genome editing in B. subtilis using RMGE- mcrA. (a) Schematic illustration of the standard operating procedure for genetic manipulation in B. subtilis. UHA and DHA represent the up- and downstream homologous arms, respectively. The asterisks indicate a deletion or replacement. (b) Detection of the amyE gene deletion using starch plates. The strains were cultured on LB plates containing 1% starch for 12 h, and then the plate was stained with iodine. W168 was used as a control. (c) Flow cytometry analysis of the expression of GFP in BS237 (W168upp::P43-gfpmut3a). 147x121mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Genome editing in S. cerevisiae using RMGE- mcrA. (a) Screening for an effective REase to regulate cell growth and death in S. cerevisiae. The strain DAY414 containing pYES2.0/CT was used as a control. (b) Construction of the plasmid pWYE3026. The plasmid pWYE3026 was generated by removing the 2µ gene fragment from pWYE3023. (c) Detection of the CAN1 gene using synthetic complete medium plates with 60 µg/mL L-canavanine. DAY414 was used as a control. 98x54mm (300 x 300 DPI)


Page 34 of 34

A novel tool for microbial genome editing using the ... - ACS Publications

Recommend Documents