Incorporation of a Synthetic Amino Acid into dCas9 Improves Control

Jan 22, 2019 - Site-Specific Chemoselective Pyrrolysine Analogues Incorporation Using the Cell-Free Protein Synthesis System. ACS Synthetic Biology...
0 downloads 0 Views 614KB Size
Subscriber access provided by EDINBURGH UNIVERSITY LIBRARY | @ http://www.lib.ed.ac.uk

Letter

Incorporation of a synthetic amino acid into dCas9 improves control of gene silencing Balwina Koopal, Aleksander J. Kruis, Nico J. Claassens, Franklin Luzia Nobrega, and John Van Der Oost ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 23, 2019

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 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 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.

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 20 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

Incorporation of a synthetic amino acid into dCas9 improves control of gene silencing

2 3 4 5

Balwina Koopal1*, Aleksander J. Kruis1,2*, Nico J. Claassens1,3, Franklin L.Nobrega1,4 & John van der Oost1

6

*- These authors contributed equally to the work.

7 8

ORCID ID:

9

Balwina Koopal - 0000-0002-5310-7528

10

Aleksander J. Kruis -0000-0002-5279-5488

11

Nico J. Claassens -0000-0003-1593-0377

12

Franklin L. Nobrega–0000-0002-8238-1083

13

John van der Oost - 0000-0001-5024-1871

14 15

Affiliations

16

1Laboratory

17

Wageningen, The Netherlands

18

2Current

19

Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

20

3Current

21

14476 Potsdam-Golm, Germany

22

4Current

23

University of Technology, Applied Sciences, Delft, The Netherlands

of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE,

address:

Bioprocess

Engineering,

Wageningen

University

and

address: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1,

address: Department of Bionanoscience – Kavli Institute of Nanoscience, Delft

24 25 26 27

Corresponding author:

28

Aleksander J. Kruis- [email protected]

ACS Paragon Plus Environment

ACS Synthetic Biology 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

29

30

Abstract

31

The CRISPR-Cas9 nuclease has been re-purposed as a tool for gene repression

32

(CRISPRi).This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either

33

initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-

34

mediated repression has been achieved with inducible promoters that regulate the expression

35

of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly

36

leaky dcas9 expression leads to significant background levels of repression of the target gene.

37

In this study, we report on the development of optimized control of dCas9-mediated silencing

38

through additional regulation at the translation level. We have introduced the TAG stop codon

39

in the dcas9 gene in order to insert a synthetic amino acid, L-biphenylalanine (BipA), at a

40

permissive site in the dCas9 protein. In the absence of BipA, a non-functional, truncated dCas9

41

is produced, but when BipA is present, the TAG codon is translated resulting in a functional,

42

full-length dCas9 protein. This synthetic, BipA-containing dCas9 variant (dCas9-BipA) could

43

still fully repress gene transcription. Comparison of silencing mediated by dCas9 to dCas9-

44

BipA revealed a 14-fold reduction in background repression by the latter system. The here

45

developed proof-of-principle system thus reduces unwanted background levels of gene

46

silencing, allowing for tight and timed control of target gene expression.

47 48 49

Keywords: CRISPR-Cas; CRISPRi; Synthetic amino acid; Cas9; Gene silencing

ACS Paragon Plus Environment

Page 2 of 20

Page 3 of 20 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

50

Gene disruption is routinely applied in the characterization of gene function and in metabolic

51

engineering strategies. However, the method is irreversible and in the case of essential genes,

52

generally lethal1. Gene silencing has been developed as an alternative which does not involve

53

the permanent removal of gene function and allows for partial and timed repression of gene

54

function. In bacteria, gene silencing has been achieved by the use of riboswitches2, repressible

55

promoters, expression of antisense RNAs3 (asRNAs) and antisense transcription4. Recently,

56

a catalytically dead version of Cas9 (dCas9) has been used to accomplish the same goal5,6.

57 58

Cas9 is the nuclease that is involved in bacterial adaptive immunity in a CRISPR type II

59

system7, which has been repurposed for other applications, notably genome editing8,9,10,11.

60

Native Streptococcus pyogenes Cas9 is able to make double-strand DNA breaks in specific

61

sequences with great precision, guided by a small CRISPR-RNA (crRNA) that has a

62

complementary sequence to the target DNA12. By replacing two catalytic residues, Asp10 and

63

His840 by Ala, a dead Cas9 (dCas9) can be generated that lost its cleavage activity but retains

64

the ability to specifically bind DNA. When dCas9 is targeted either to the promoter region

65

upstream a gene, or to the non-template strand within a gene, it prevents binding of the RNA

66

polymerase or transcription elongation, respectively. As such, dCas9 has successfully been

67

used to silence gene expression5,6, a technique that is also known as CRISPR interference

68

(CRISPRi)13. An advantage of using dCas9 for gene silencing is that any gene may be targeted

69

depending on the spacer sequence of the provided crRNA guide14. In contrast, to control gene

70

expression using an inducible promoter or a riboswitch, the upstream region of the gene of

71

interest needs to be modified, implying that adjusting the targeting specificity for these systems

72

is relatively laborious compared to dCas9 targeting. Furthermore, dCas9/guide complexes

73

have a very high affinity for a complementary DNA targets15,16 meaning that efficient gene

74

silencing can be achieved even with low levels of dCas95. In comparison, asRNAs generally

75

need to be expressed in excess compared to the target mRNA17,18,19.

76

The high binding efficiency of dCas9 can also be a drawback when used as a gene silencing

77

tool. The problem originates from the inherent leakiness of inducible promoters20 that have

78

been used to control dCas9 expression21,6. Due to promoter leakiness, there is constant, low

79

level dCas9 production in the absence of the inducer, resulting in background gene silencing.

80

This problem was partly solved when a single copy of the repression machinery was integrated

81

into the E. coli genome21. Peters et al. (2016)22 used leaky chromosomal dCas9 expression in

82

B. subtilus in a study of essential genes, which gave about 3-fold repression without induction.

83

However, for many experiments the residual gene silencing is undesired as the difference

84

before and after induced silencing will be lower and harder to study.

ACS Paragon Plus Environment

ACS Synthetic Biology 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

85

Methods to control Cas9 activity post-transcriptionally have been established in eukaryotic

86

cells23. The major goal of the conditional control has been to reduce off-target effects of

87

genome editing24,25. For example, Cas9 variants, containing self-excising inteins (inducing re-

88

activation of Cas9), or regulated degradation tags (inducing rapid degradation of Cas9 in the

89

absence of a synthetic ligand) have been engineered26,27. Cas9 has also been split into two

90

parts that are joined to form a functional protein after stimulation by an external ligand28 or blue

91

light29,30. Attempts to use post-transcriptional control of dCas9 activity in E. coli are limited. One

92

example is a 4-hydroxytamoxifen-dependent dCas9 that was developed with a high throughput

93

screening system in E. coli. While 4-hydroxytamoxifen-dependent gene silencing was

94

achieved, there still was some residual gene silencing in the absence of the inducer31. Other

95

studies in E. coli corrected for leaky dCas9 expression by introducing mismatches into the

96

sgRNA, thereby weakening the interaction with the DNA. As a result, dCas9 became inefficient

97

at silencing genes at low protein levels. The latter approach may need to be optimised per

98

target gene and sgRNA binding site5. To circumvent this, the red fluorescent protein-encoding

99

rfp gene was fused to the 5’-end of an essential gene, and silenced with a tested set of sgRNAs

100

with different degrees of mismatches, which enabled tuning of precise and non-leaky gene

101

repression32,33. However, the rfp fusions are themselves laborious, and may affect gene

102

expression and function. Alternatively, improved control over dCas9 repression in E. coli was

103

achieved by expressing asRNA to silence sgRNAs, but this also relies on optimization of the

104

designed asRNAs34. In the cyanobacterium Synechcocystis sp. PCC 6083, tighter control over

105

dCas9-mediated silencing was achieved by conditional expression of both cas9 and sgRNA

106

genes35.

107

In this study we improved post-transcriptional control over dCas9-mediated gene silencing in

108

E. coli through introduction of a novel regulatory mechanism at the level of translation, thereby

109

making dCas9 production dependent on supplementation of a synthetic amino acid, L-

110

biphenylalanine (BipA).

111 112

ACS Paragon Plus Environment

Page 4 of 20

Page 5 of 20 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

113

We first established a baseline for further experiments by using (standard) dCas9 controlled

114

by an inducible promoter to silence the expression of the mrfp gene. We chose two promoters

115

that were reported to give relatively tight control over gene expression: the anhydrotetracyclin

116

(aTc)-inducible PLtetO-1 promoter system36,6, and the m-toluate inducible Pm promoter

117

system37,38. A CRISPR array containing two spacers that were previously shown to effectively

118

silence mrfp expression6 (Supplementary Figure 1) was expressed constitutively. The two

119

spacer spacers were not predicted to have any off-target effects39. They also did not contain

120

sequences that could lead to a bad-seed effect40. For both promoters we observed substantial

121

silencing of mrfp in the absence of the inducer compared to the non-targeting control condition

122

without a CRISPR array (Pm promoter: -84% ± 1% of mRFP/OD600; PLtetO-1 promoter: >-99%

123

± 0% of mRFP/OD600, Figure 1A; see also Supplementary Figure 2-3A), suggesting that neither

124

of the tight promoters is suitable enough for conditional dCas9-mediated gene silencing. We

125

aimed to improve this system by adding a translational level of control.

126 127

In the here described system, the synthetic amino acid BipA is incorporated in dCas9 through

128

translation of the TAG codon. Commonly, the TAG codon is a stop codon that is recognized

129

by release factor 1, which terminates translation41. However, in E. coli C321.ΔA.exp42, all TAG

130

stop codons have been replaced by TAA, and release factor 1 has been disrupted. The TAG

131

codon was repurposed to code for BipA by providing the appropriate translation machinery: a

132

tRNA that recognizes the TAG codon (tRNACUA), and an aminoacyl synthetase (aaRS) that

133

charges the tRNACUA with BipA43,44 (Figure 2A). To investigate whether BipA-dependent

134

translation of the TAG codon can serve as a better control mechanism over dCas9-mediated

135

gene silencing, we substituted the cas9-GAT30 codon, encoding Asp10, with the TAG codon.

136

We also included a His840Ala substitution to completely abolish the cleavage activity of Cas9.

137

In the presence of the orthogonal translation machinery, a new version of synthetic dCas9 is

138

produced, which has BipA incorporated instead of the catalytic Asp10: dCas9-BipA. However,

139

this only happens when BipA is supplied to the growth medium (Figure 2A). In the absence of

140

BipA, the TAG codon should not be translated and dCas9 should not be produced (Figure 2B).

141

Thus, withdrawal of BipA from the growth medium should provide control over dCas9-mediated

142

gene silencing, using E. coli C321.ΔA.exp as a chassis.

ACS Paragon Plus Environment

ACS Synthetic Biology 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

143 144

Figure 1. Controlling dCas9-mediated mrfp silencing using either inducible promoters or

145

conditional translation of the TAG codon. A – E. coli (Pm-dCas9) and E. coli (PLtetO-1-dCas9) grown

146

with and without m-toluate or aTc, respectively. B – E. coli (Pspy-dCas9-BipA) grown with and without

147

BipA supplementation. Fluorescence and OD600were measured after 7 hours of growth when the cells

148

were in the exponential growth phase. Shown are the mRFP/OD600 values, error bars indicate standard

149

error of biological duplicates, measured as technical triplicates.

150 151

To test whether dCas9-BipA is still able to silence gene expression, we expressed dCas9-BipA

152

under the native, constitutive S. pyogenes promoter5. Figure 1B shows that E. coli(Pspy-

153

dCas9-BipA) cells were able to silence mrfp expression almost completely when a CRISPR

154

array was present, but only when BipA was supplemented (>-99% ± 0% of mRFP/OD600

155

compared to no array; see also Supplementary figure 4A). Since BipA is considerably larger

156

and more hydrophobic compared to the negatively charged Asp, it is unlikely that it could

157

restore dCas9 cleaving activity. In fact, we did not observe a growth defect in antibiotic-

158

containing medium when both BipA and the CRISPR array were present (Supplementary

159

Figure 4B), which indicates that dCas9-BipA did not cleave the plasmid encoding the antibiotic

160

marker and mrpf. This result shows that translational control of dCas9-BipA may serve as an

161

alternative or addition to transcriptional control. However, we still observed some mrfp

162

silencing without supplementation of BipA (-35% ± 1% of mRFP/OD600 compared to no array;

163

Figure 1B). This can be explained by non-specific activity of the aaRS/tRNACUA-pair43. When

164

BipA is not available, the aaRS may charge the tRNACUA with a native amino acid. This is likely

165

Tyr, as the aaRS used in this study is derived from an aaRS that charges the tRNACUA with

166

Tyr. Both transcriptional and translational control of dCas9-BipA production thus showed a

167

certain degree of leakiness.

ACS Paragon Plus Environment

Page 6 of 20

Page 7 of 20 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

168

To overcome this, we combined BipA-dependent dCas9-BipA production with inducible dcas9-

169

bipA expression under the control of either the PLtetO-1 or the Pm promoter. This resulted in

170

optimal control over mRFP production. Adding both the inducer and BipA to our system

171

resulted in virtually complete mrfp silencing for both the Pm promoter (-97% ± 1% of

172 173

Figure 2. Combined transcriptional and translational control of dCas9-mediated gene silencing.

174

The TAG codon is inserted in the dcas9 gene and can be translated to incorporate L-biphenylalanine

175

(BipA) when the suitable translation machinery is provided: a tRNACUA that recognizes the UAG codon

176

and an aminoacyl-synthetase (aaRS) that charges the tRNA with BipA. To facilitate translation of the

177

TAG codon, E. coli C321.ΔA.exp42 is used as a chassis. An inducible promoter is used to drive dCas9

178

expression as an additional control mechanism. A – Without gene inducer or BipA, no dCas9 or mRFPis

179

produced. B –When both BipA and the inducer are provided, dCas9-BipA is produced, which silences

180

mRFP production. C – Constructs used in this study.

181

mRFP/OD600 relative to no array) and the PLtetO-1 promoter (-98% ± 0% of mRFP/OD600

182

compared to no array) (Figure 3; see also Supplementary Figures 5-6). In the absence of the

183

inducer and BipA, we observed little mrfp silencing when the Pm promoter was used (-6% ±

184

3% of mRFP/OD600 compared to no array). Changing to the PLtetO-1 promoter also led to an

185

improvement of our system, although there was more mrfp silencing in the absence of the

186

inducer and BipA (-16 ± 2% of mRFP/OD600 relative to no array) compared to the Pm promoter.

ACS Paragon Plus Environment

ACS Synthetic Biology 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

187

In fact, thePLtetO-1 promoter did not require induction to fully silence mrfp in the presence of

188

BipA (Figure 3). This indicates that the PLtetO-1 promoter is more leaky than the Pm promoter,

189

which is in agreement with our previous results (Figure 1A).

190

Surprisingly, supplementation with m-toluate, aTc or BipA lowered mRFP production roughly

191

10-20% in E. coli strains when no CRISPR array was present (compared to non-supplemented

192

controls). When both an inducer and BipA were supplied, the reduction in mRFP was additive.

193

This unspecific effect was observed in both dCas9 and dCas9-BipA producing E. coli strains

194

(Supplementary Table 1). In the case of m-toluate and aTc, it is possible that the additional

195

burden of dCas9 production diverted cellular resources from mRFP production. Furthermore,

196

it was shown that high dCas9 production in E. coli causes growth defects and other changes45,

197

which is in line with our observations (Supplementary Figures 2B-6B). This might be alleviated

198

by lowering dCas9 expression, which should still preserve the strong repression33. However,

199

the observed slight mRFP repression by BipA in the regular dCas9 strain cannot be explained

200

by increased dCas9 (Supplementary Table 1). BipA did not have negative effect on E. coli

201

growth (Supplementary Figures 2B-6B), demonstrating it is unlikely that the reduced mRFP

202

production is due to BipA toxicity. Actually, strains producing regular dCas9 cultivated in the

203

presence of BipA grew a bit faster for unknown reasons. These small increases in OD600 can

204

significantly impact mRFP/OD600 values and may explain the reduced mRFP/OD600 production.

ACS Paragon Plus Environment

Page 8 of 20

Page 9 of 20 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

205 206

Figure 3. Combining conditional translation of the TAG codon and an inducible promoter

207

provides optimal control over dCas9-mediated mrfp silencing. A - E. coli (Pm-dCas9-BipA) and B

208

- E. coli (PLtetO-1-dCas9-BipA) cells were grown in the absence or presence of BipA and/or the inducer.

209

Fluorescence and OD600 were measured after 7 hours of growth when cells were in the exponential

210

growth phase. Shown are the mRFP/OD600 values, error bars indicate standard error of biological

211

duplicates, measured as technical triplicates.

212

Combining transcriptional and translational control over dCas9-BipA production improved

213

control over the system, with virtually complete repression when induced, and minimal

214

leakiness in the absence of both inducer and BipA. However, the experiments have so far

215

focused only on situations where all components were present from the beginning. In practice,

216

it may be desirable to regulate gene expression in time. We therefore tested whether mRFP

217

production could be stopped in actively growing of E. coli(Pm-dCas9-BipA) cultures. BipA and

218

m-toluate were supplemented four hours after inoculation. We observed that the mRFP/OD600

219

started decreasing approximately one hour after BipA and m-toluate were added. After 13

220

hours, the mRFP/OD600 dropped back to its initial value (Figure 4A). Overall, the results of this

ACS Paragon Plus Environment

ACS Synthetic Biology 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

221

experiment match the observations in Figure 3. mRFP production was completely diminished

222

after adding m-toluate and BipA, and partially repressed when only one of the components

223

was present. Interestingly, for the negative control mRFP production was stable for a short

224

time, shortly after the other samples were induced (to the negative control only solvents were

225

added). This period coincides with the exponential phase of growth. The exponential phase

226

was also observed to give temporary stable mRFP/OD600 in other experiments with induction

227

from the start (Supplementary Figures 2A-6A). We also observed that the cultures were

228

growing rather slowly in general. This was probably caused by the cost of maintaining three

229

plasmids, overexpressing multiple proteins (including dCas9) as well as poor aeration from

230

growing in the microplatereader46. Regardless, our results emphasize that timed control of

231

mRFP production could be achieved by supplementing BipA and m-toluate to the system.

232 233

Figure 4. dCas9-BipA allows for temporal control of mRFP production in actively growing cells.

234

E. coli(Pm-dCas9-BipA) was cultivated in LB medium. After 4 hours, m-toluate, BipA or their solvents

235

were added (black arrow). Fluorescence and OD600were monitored over time. The values represent

236

averages of biological duplicates, measured as technical triplicates. All replicates showed the same

237

trends. Error bars were omitted to improve figure clarity but are available in Supplementary Figure 7. A

238

– mRFP/OD600values observed during 14 hours of cultivation. B – OD600 values observed during 14

239

hours of cultivation.

240

In this study, we demonstrated a new system for inducible gene silencing using a novel,

241

synthetic form of dCas9, which has the synthetic amino acid BipA incorporated: dCas9-BipA.

242

The novel dCas9 variant retains the ability to silence gene expression and is dependent on

243

BipA supplementation. Thus, it may serve as an alternative to transcription-controlled dCas9-

ACS Paragon Plus Environment

Page 10 of 20

Page 11 of 20 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

244

expression. However, the BipA dependency is leaky; without supplementation of BipA to the

245

medium, there is still some gene silencing. When translational control over dCas9 production

246

was combined with transcriptional control using an inducible promoter, the leakiness was

247

reduced significantly. We observed that pairing BipA-dependency with the Pm promoter gave

248

the best results. It greatly reduced background gene silencing while enabling almost complete

249

silencing when the system was fully induced. We used mrfp to demonstrate the usefulness of

250

the system, but in principle any gene (in this E. coli strain) can be targeted. This may be

251

particularly facilitated by the novel Cas9 versions that have been developed with different PAM

252

preferences47,48,49,50.

253

Alternative solutions based on post-transcriptional (d)Cas9 control have focused on reducing

254

Cas9 off-target cleavage in eukaryotic cells. Cas9 has been engineered to switch between an

255

inactive and an active state by means of external stimuli, such as small ligands or blue light.

256

An advantage of these systems is that the response to the stimulus may be faster since Cas9

257

does not need to be synthesised de novo. However, most of the conditional Cas9 variants

258

retain at least some level of cleavage activity in their “inactive” form51,24,26. If these concepts

259

were applied in E. coli for dCas9-mediated gene silencing, the residual activity may cause

260

unwanted repression, as opposed to the system developed in this study. On the other hand,

261

there may be opportunities to combine transcriptional and post-transcriptional control as well

262

with these systems. Another demonstrated approach to prevent unwanted repression by

263

dCas9 is to control both dcas9 and sgRNA expression by two different inducible promoters35.

264

The here described system relies on a genetically recoded strain for efficient translation of the

265

TAG codon, which may be considered a drawback for general applicability. However, other

266

orthogonal aaRS/tRNACUA-pairs, recognizing for example non-natural 4-base-frameshift

267

codons52, could be used to introduce BipA, or other unnatural amino acids, into dCas9. This

268

would potentially allow the system to be used in any E. coli strain. Furthermore, our design

269

may also find applications in controlling the activity/expression of other Cas9 versions and

270

even other proteins.

271

ACS Paragon Plus Environment

ACS Synthetic Biology 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

272

Methods

273

Plasmids and strains

274

Table 1. Plasmids used in this study. Plasmid

Use (antibiotic)

Page 12 of 20

Source

pdCas9 (Addgene plasmid # 46569) Construction of pET26B-dCas9 Bikard et al. (2013)5 variants (Cam) pET26B

Construction of pET26B-dCas9 Novagen variants (Kan)

pET26B-dCas9-GCT10TAG

mrfp silencing (Kan)

This study

pET26B-dCas9-GCT10TAG-

mrfp silencing (Kan)

This study

mrfp silencing (Kan)

This study

-dCas9- mrfp silencing (Kan)

This study

RFParray pET26B-XylS-Pm-dCas9 pET26B-XylS-Pm GCT10TAG pET26B-XylS-Pm-dCas9-RFParray

mrfp silencing (Kan)

This study

pET26B-XylS-Pm-dCas9-

mrfp silencing (Kan)

This study

pET26B- PLtetO-1-dCas9

mrfp silencing (Kan)

This study

pET26B-PLtetO-1-dCas9-

mrfp silencing (Kan)

This study

pET26B- PLtetO-1-dCas9-RFParray mrfp silencing (Kan)

This study

pET26B-PLtetO-1-dCas9-

This study

GCT10TAG-RFParray

GCT10TAG mrfp silencing (Kan)

GCT10TAG-RFParray pEVOL-BipA

Translation of the TAG codon Mandell et al. (2015)44 (Cam)

pBbS5a-RFP (Addgene plasmid # Reporter for mrfp silencing (Amp) Lee et al. (2011)53 35283) 275 276

ACS Paragon Plus Environment

Page 13 of 20 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

277 278

ACS Synthetic Biology

Table 2. E. coli strains used in this study. Negative controls used lack the CRISPR-array for targeting mrfp. E.coli strain Strain genotype Source C321.ΔA.exp(Addgene

E. coli MG1655 Δ(ybhB-bioAB)::zeoRΔprfA; Lajoie

et

strain #49018)

all 321 TAG codons changed to TAA

XylS-Pm-dCas9

E. coli C321.ΔA.exp carrying: pET26B- XylS- This study

al.

(2013)42

Pm-dCas9-RFParray, pEVOL-BipA, pBbS5a-RFP PLtetO-1-dCas9

E.

coli

C321.ΔA.exp

carrying:

PLtetO-1-dCas9-RFParray,

pET26B- This study

pEVOL-BipA,

pBbS5a-RFP dCas9-BipA

E.

coli

C321.ΔA.exp

carrying:

pET26B- This study

dCas9-GCT10TAG-RFParray, pEVOL-BipA, pBbS5a-RFP XylS-Pm-dCas9-BipA

E. coli C321.ΔA.exp carrying: pET26B-XylS- This study Pm-dCas9-RFParray, pEVOL-BipA, pBbS5aRFP

PLtetO-1-dCas9-BipA

E.

coli

C321.ΔA.exp

carrying:

PLtetO-1-dCas9-RFParray,

pET26B- This study

pEVOL-BipA,

pBbS5a-RFP 279 280

Strain and plasmid construction

281

E. coli C321.ΔA.exp42 and adk.d6_tyrS.d844 were received from Prof. George Church (Harvard

282

University, Boston), and were used to perform gene silencing experiments and to obtain

283

pEVOL-BipA, respectively. PCR reactions were performed using Q5 polymerase (NEB).

284

Fragments were either purified using the Zymo PCR cleanup kit or excised from gel and

285

purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey Nagel). Ligation was

286

performed using T4 ligase (Thermo Fisher) and restriction-digestions were performed using

287

NEB restriction enzymes in their accompanying buffers. Colony PCR was performed using

288

OneTaq polymerase (NEB), followed by Miniprep using the GeneJET Plasmid Miniprep Kit

289

(Thermo Fisher). Candidate clones were verified by Sanger sequencing.

290

pET26B-dCas9 was constructed using restriction/digestion cloning, using parts of

291

pdCas95 and pET26B (Novagen). To make mutations in dCas9, mutagenesis PCR was used

292

based on the Agilent Quickchange protocol. A CRISPR array of two spacers targeting the non-

293

template strand of mrfp6 was inserted in pET26B-dCas9 and pET26B-dCas9-GCT10TAG by

294

annealing two complementary oligonucleotides (Supplementary Table 3) and cloning them

ACS Paragon Plus Environment

ACS Synthetic Biology 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

295

using a Golden-Gate-like protocol54. The constitutive dCas9 promoter was replaced by either

296

the Pm37 (obtained from pHH100) or the PLtetO-1 promoter (as was used by Qi et al. (2013)6,

297

ordered as a gBlock from IDT). The vectors were assembled by Gibson assembly using the

298

Hifi DNA assembly cloning kit (NEB). Assembled plasmids were either transformed into

299

commercial chemically competent DH5α cells (NEB) according to the manufacturers protocol.

300

E. coli strain C321.ΔA.exp was transformed according to the protocol described in Lajoie et al.

301

(2013)42.

302 303

Strain cultivation and fluorescence assays

304

E. coli strains were routinely cultured in LB, supplemented with, chloramphenicol (25 ug/mL),

305

kanamycin (50 ug/mL), ampicillin (50 ug/mL) andbleomycin (5 ug/mL), where appropriate.

306

Gene silencing experiments were performed by cultivating strains in 96-well plates (Greiner)

307

in a SynergyMx platereader (BioTek), at 37°C while shaking continuously. OD600 and mRFP

308

fluorescence (excitation: 584nm, emission: 607nm) were measured every 30 min and 2 µL

309

overnight precultures (in LB) were inoculated into 200µL LB, supplemented with IPTG (100

310

µM), BipA (Fluorochem; 200uM), anhydrotetracycline (aTc; 1 µM) or m-toluate (2 mM), where

311

appropriate. Alternatively, BipA and aTc or m-toluate were added after 4 hours of growth in the

312

same concentrations.

313

314

Acknowledgements

315

We would like to thank Prof. G. Church for his kind donation of E. coli C321.ΔA.exp (Addgene

316

strain #49018), E. coli adk.d6_tyrS.d8 and plasmid pEVOL-BipA. We are grateful to Dr. R. Lale

317

for plasmid pHH100 and to Prof. J.D. Keasling for plasmid pBbS5a-RFP. pdCas9 was a gift

318

from Luciano Marraffini (Addgene plasmid #46569). Lastly, we would like the Wageningen

319

iGEM team 2016 for their support.

320

J.v.d.O. is supported by the Netherlands Organization for Scientific Research (NWO) through

321

a TOP grant (714.015.001) and a Gravitation grant (024.003.019).

322 323 324

ACS Paragon Plus Environment

Page 14 of 20

Page 15 of 20 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

325

Supporting information.

326

Additional figures and tables.

327

Supplementary methods.

328 329

ACS Paragon Plus Environment

ACS Synthetic Biology 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

330

Page 16 of 20

References

331 332

(1)

Hutchison III, C. a.; Peterson, S. N.; Gill, S. R.; Cline, R. T.; White, O.; Fraser, C. M.;

333

Smith, H. O.; Venter, J. C. Global Transposon Mutagenesis and a Minimal Mycoplasma

334

Genome.

335

https://doi.org/10.1126/science.286.5447.2165.

336

(2)

Science

(80-.

).

1999,

286

(5447),

2165–2169.

Muranaka, N.; Abe, K.; Yokobayashi, Y. Mechanism‐Guided Library Design and Dual

337

Genetic Selection of Synthetic OFF Riboswitches. ChemBioChem 2009, 10 (14), 2375–

338

2381.

339

(3)

Nakashima, N.; Tamura, T. Gene Silencing in Escherichia Coli Using Antisense RNAs

340

Expressed from Doxycycline-Inducible Vectors. Lett. Appl. Microbiol. 2013, 56 (6), 436–

341

442. https://doi.org/10.1111/lam.12066.

342

(4)

343 344

Brophy, J. A.; Voigt, C. A. Antisense Transcription as a Tool to Tune Gene Expression. Mol. Syst. Biol. 2016, 12 (1), 854–854. https://doi.org/10.15252/msb.20156540.

(5)

Bikard, D.; Jiang, W.; Samai, P.; Hochschild, A.; Zhang, F.; Marraffini, L. A.

345

Programmable Repression and Activation of Bacterial Gene Expression Using an

346

Engineered CRISPR-Cas System. Nucleic Acids Res. 2013, 41 (15), 7429–7437.

347

https://doi.org/10.1093/nar/gkt520.

348

(6)

Qi, L. S.; Larson, M. H.; Gilbert, L. A.; Doudna, J. A.; Weissman, J. S.; Arkin, A. P.; Lim,

349

W. A. Repurposing CRISPR as an RNA-Γuided Platform for Sequence-Specific Control

350

of

351

https://doi.org/10.1016/j.cell.2013.02.022.

352

(7)

Gene

Expression.

Tools

354

https://doi.org/10.1002/bit.25851. (8)

2013,

152

(5),

1173–1183.

Luo, M. L.; Leenay, R. T.; Beisel, C. L. Current and Future Prospects for CRISPR-Based

353 355

Cell

in

Bacteria.

Biotechnol.

Bioeng.

2016,

113

(5),

930–943.

Jiang, W.; Bikard, D.; Cox, D.; Zhang, F.; Marraffini, L. A. RNA-Guided Editing of

356

Bacterial Genomes Using CRISPR-Cas Systems. Nat. Biotechnol. 2013, 31 (3), 233–

357

239. https://doi.org/10.1038/nbt.2508.

358

(9)

Cong, L.; Ran, A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang,

359

W.; Marraffini, L.; et al. Multiplex Genome Engineering Using CRISPR/Cas Systems.

360

Science (80-. ). 2013, 339 (6121), 819–823. https://doi.org/10.1038/nbt1319.

361

(10)

Li, J.; Aach, J.; Norville, J. E.; Mccormack, M.; Bush, J.; Church, G. M.; Sheen, J.

362

Multiplex and Homologous Recombination-Mediated Genome Editing in Arabidopsis

363

and Nicotianabenthamiana Using Guide RNA and Cas9. Nat. Biotechnol. 2013, 31 (8),

364

688–691. https://doi.org/10.1038/nbt.2654.Multiplex.

365 366

(11)

Wang, H.; La Russa, M.; Qi, L. S. CRISPR/Cas9 in Genome Editing and Beyond. Annu. Rev. Biochem. 2016, 85 (1), 227–264. https://doi.org/10.1146/annurev-biochem-

ACS Paragon Plus Environment

Page 17 of 20 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

367 368

060815-014607. (12)

Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. A

369

Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.

370

Science (80-. ). 2012, 337 (August), 816–822. https://doi.org/10.1126/science.1225829.

371

(13)

Larson, M. H.; Gilbert, L. A.; Wang, X.; Lim, W. A.; Weissman, J. S.; Qi, L. S. CRISPR

372

Interference (CRISPRi) for Sequence-Specific Control of Gene Expression. Nat. Protoc.

373

2013, 8 (11), 2180–2196. https://doi.org/10.1038/nprot.2013.132.

374

(14)

Dominguez, A. A.; Lim, W. A.; Qi, L. S. Beyond Editing: Repurposing CRISPR-Cas9 for

375

Precision Genome Regulation and Interrogation. Nat. Rev. Mol. Cell Biol. 2016, 17 (1),

376

5–15. https://doi.org/10.1038/nrm.2015.2.

377

(15)

Sternberg, S. H.; Redding, S.; Jinek, M.; Greene, E. C.; Doudna, J. A. DNA Interrogation

378

by the CRISPR RNA-Guided Endonuclease Cas9. Nature 2014, 507 (7490), 62–67.

379

https://doi.org/10.1038/nature13011.

380

(16)

Knight, S. C.; Xie, L.; Deng, W.; Guglielmi, B.; Witkowsky, L. B.; Bosanac, L.; Zhang, E.

381

T.; Beheiry, M. E.; Masson, J. B.; Dahan, M.; et al. Dynamics of CRISPR-Cas9 Genome

382

Interrogation in Living Cells. Science (80-. ). 2015, 350 (6262), 823–826.

383

https://doi.org/10.1126/science.aac6572.

384

(17)

385 386

Nakashima, N.; Miyazaki, K. Bacterial Cellular Engineering by Genome Editing and Gene Silencing. Int. J. Mol. Sci. 2014, 15 (2), 2771–2793.

(18)

Nakashima, N.; Tamura, T.; Good, L. Paired Termini Stabilize Antisense RNAs and

387

Enhance Conditional Gene Silencing in Escherichia Coli. Nucleic Acids Res. 2006, 34

388

(20). https://doi.org/10.1093/nar/gkl697.

389

(19)

Chen, H.; Ferbeyre, G.; Cedergren, R. Efficient Hammerhead Ribozyme and Antisense

390

RNA Targeting in a Slow Ribosome Escherichia Coli Mutant. Nat. Biotechnol. 1997, 15

391

(5), 432–453.

392

(20)

393 394

Rosano, G. L.; Ceccarelli, E. A. Recombinant Protein Expression in Escherichia Coli: Advances and Challenges. Front. Microbiol. 2014, 5, 1–17.

(21)

Li, X. T.; Jun, Y.; Erickstad, M. J.; Brown, S. D.; Parks, A.; Court, D. L.; Jun, S.

395

TCRISPRi: Tunable and Reversible, One-Step Control of Gene Expression. Sci. Rep.

396

2016, 6, 1–12. https://doi.org/10.1038/srep39076.

397

(22)

Peters, J. M.; Colavin, A.; Shi, H.; Czarny, T. L.; Larson, M. H.; Wong, S.; Hawkins, J.

398

S.; Lu, C. H. S.; Koo, B. M.; Marta, E.; et al. A Comprehensive, CRISPR-Based

399

Functional Analysis of Essential Genes in Bacteria. Cell 2016, 165 (6), 1493–1506.

400

https://doi.org/10.1016/j.cell.2016.05.003.

401

(23)

Wu, W. Y.; Lebbink, J. H. G.; Kanaar, R.; Geijsen, N.; Van Der Oost, J. Genome Editing

402

by Natural and Engineered CRISPR-Associated Nucleases. Nat. Chem. Biol. 2018, 14

403

(7), 642–651. https://doi.org/10.1038/s41589-018-0080-x.

ACS Paragon Plus Environment

ACS Synthetic Biology 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

404

(24)

405 406

Page 18 of 20

Zhou, W.; Deiters, A. Conditional Control of CRISPR/Cas9 Function. Angew. Chemie 2016, 55 (18), 5394–5399.

(25)

Guha, T. K.; Wai, A.; Hausner, G. Programmable Genome Editing Tools and Their

407

Regulation for Efficient Genome Engineering. Comput. Struct. Biotechnol. J. 2017, 15,

408

146–160. https://doi.org/10.1016/j.csbj.2016.12.006.

409

(26)

Davis, K. M.; Pattanayak, V.; Thompson, D. B.; Zuris, J. A.; Liu, D. R. Small Molecule-

410

Triggered Cas9 Protein with Improved Genome- Editing Specificity. Nat. Chem. Biol.

411

2015, 11 (5), 316–318. https://doi.org/10.1038/nchembio.1793.Small.

412

(27)

Senturk, S.; Shirole, N. H.; Nowak, D. G.; Corbo, V.; Pal, D.; Vaughan, A.; Tuveson, D.

413

A.; Trotman, L. C.; Kinney, J. B.; Sordella, R. Rapid and Tunable Method to Temporally

414

Control Gene Editing Based on Conditional Cas9 Stabilization. Nat. Commun. 2017, 8

415

(May 2015), 1–10. https://doi.org/10.1038/ncomms14370.

416

(28)

Zetsche, B.; Volz, S. E.; Zhang, F. A Split Cas9 Architecture for Inducible Genome

417

Editing and Transcription Modulation. Nat. Biotechnol. 2015, 2 (2), 147–185.

418

https://doi.org/10.1515/jci-2013-0007.Targeted.

419

(29)

Polstein, L. R.; Gersbach, C. A.; Carolina, N.; States, U.; Biology, C.; Carolina, N.;

420

Carolina, N. A Light-Inducible CRISPR-Cas9 System for Control of Endogenous Gene

421

Activation. 2015, 11 (3), 198–200. https://doi.org/10.1038/nchembio.1753.A.

422

(30)

Nihongaki, Y.; Yamamoto, S.; Kawano, F.; Suzuki, H.; Sato, M. CRISPR-Cas9-Based

423

Photoactivatable Transcription System. Chem. Biol. 2015, 22 (2), 169–174.

424

https://doi.org/10.1016/j.chembiol.2014.12.011.

425

(31)

Oakes, B. L.; Nadler, D. C.; Flamholz, A.; Fellmann, C.; Staahl, B. T.; Doudna, J. A.;

426

Savage, D. F. Profiling of Engineering Hotspots Identifies an Allosteric CRISPR-Cas9

427

Switch. 2016, 34 (6), 646–651. https://doi.org/10.1038/nbt.3528.Profiling.

428

(32)

Vigouroux, A.; Oldewurtel, E.; Cui, L.; Teeffelen, S. van; Bikard, D. Engineered CRISPR-

429

Cas9 System Enables Noiseless, Fine-Tuned and Multiplexed Repression of Bacterial

430

Genes. bioRxiv 2017, 164384. https://doi.org/10.1101/164384.

431

(33)

Vigouroux, A.; Oldewurtel, E.; Cui, L.; Bikard, D.; van Teeffelen, S. Tuning DCas9’s

432

Ability to Block Transcription Enables Robust, Noiseless Knockdown of Bacterial Genes.

433

Mol. Syst. Biol. 2018, 14 (3), e7899. https://doi.org/10.15252/msb.20177899.

434

(34)

Lee, Y. J.; Hoynes-O’Connor, A.; Leong, M. C.; Moon, T. S. Programmable Control of

435

Bacterial Gene Expression with the Combined CRISPR and Antisense RNA System.

436

Nucleic Acids Res. 2016, 44 (5), 2462–2473. https://doi.org/10.1093/nar/gkw056.

437

(35)

Yao, L.; Cengic, I.; Anfelt, J.; Hudson, E. P. Multiple Gene Repression in Cyanobacteria

438

Using

439

https://doi.org/10.1021/acssynbio.5b00264.

440

(36)

CRISPRi.

ACS

Synth.

Biol.

2016,

5

(3),

207–212.

Lutz, R.; Bujard, H. Independent and Tight Regulation of Transcriptional Units in

ACS Paragon Plus Environment

Page 19 of 20 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

441

Escherichia Coli via the LacR/O, the TetR/O and AraC/I1-I2regulatory Elements. Nucleic

442

Acids Res. 1997, 25 (6), 1203–1210. https://doi.org/10.1093/nar/25.6.1203.

443

(37)

Lale, R.; Berg, L.; Stuttgen, F.; Netzer, R.; Stafsnes, M.; Brautaset, T.; Aune, T. E. V.;

444

Valla, S. Continuous Control of the Flow in Biochemical Pathways through 5’

445

Untranslated Region Sequence Modifications in MRNA Expressed from the Broad-Host-

446

Range Promoter Pm. Appl. Environ. Microbiol. 2011, 77 (8), 2648–2655.

447

https://doi.org/10.1128/AEM.02091-10.

448

(38)

Balzer, S.; Kucharova, V.; Megerle, J.; Lale, R.; Brautaset, T.; Valla, S. A Comparative

449

Analysis of the Properties of Regulated Promoter Systems Commonly Used for

450

Recombinant Gene Expression in Escherichia Coli. Microb. Cell Fact. 2013, 12 (1), 1–

451

14. https://doi.org/10.1186/1475-2859-12-26.

452

(39)

Labun, K.; Montague, T. G.; Gagnon, J. A.; Thyme, S. B.; Valen, E. CHOPCHOP v2: A

453

Web Tool for the next Generation of CRISPR Genome Engineering. Nucleic Acids Res.

454

2016, 44 (W1), W272–W276. https://doi.org/10.1093/nar/gkw398.

455

(40)

Cui, L.; Vigouroux, A.; Rousset, F.; Varet, H.; Khanna, V.; Bikard, D. A CRISPRi Screen

456

in E. Coli Reveals Sequence-Specific Toxicity of DCas9. Nat. Commun. 2018.

457

https://doi.org/10.1038/s41467-018-04209-5.

458

(41)

Scolnick, E.; Tompkins, R.; Caskey, T.; Nirenberg, M. Release Factors Differing in

459

Specificity for Terminator Codons. Proc. Natl. Acad. Sci. U. S. A. 1968, 61 (2), 768–774.

460

https://doi.org/10.1073/pnas.61.2.768.

461

(42)

Lajoie, M. J.; Rovner, A. J.; Goodman, D. B.; Aerni, H.; Haimovich, A. D.; Kuznetsov,

462

G.; Mercer, J. a; Wang, H. H.; Carr, P. a; Mosberg, J. a; et al. Genomically Recoded

463

Organisms Expand Biological Functions. Science 2013, 342 (October), 357–360.

464

https://doi.org/10.1126/science.1241459.

465

(43)

Young, T. S.; Ahmad, I.; Yin, J. A.; Schultz, P. G. An Enhanced System for Unnatural

466

Amino Acid Mutagenesis in E. Coli. J. Mol. Biol. 2010, 395 (2), 361–374.

467

https://doi.org/10.1016/j.jmb.2009.10.030.

468

(44)

Mandell, D. J.; Lajoie, M. J.; Mee, M. T.; Takeuchi, R.; Kuznetsov, G.; Norville, J. E.;

469

Gregg, C. J.; Stoddard, B. L.; Church, G. M. Biocontainment of Genetically Modified

470

Organisms by Synthetic Protein Design. Nature 2015, 518 (7537), 55–60.

471

https://doi.org/10.1038/nature14121.Biocontainment.

472

(45)

Cho, S.; Choe, D.; Lee, E.; Kim, S. C.; Palsson, B.; Cho, B. K. High-Level DCas9

473

Expression Induces Abnormal Cell Morphology in Escherichia Coli. ACS Synth. Biol.

474

2018, 7 (4), 1085–1094. https://doi.org/10.1021/acssynbio.7b00462.

475

(46)

Chavez, M.; Ho, J.; Tan, C. Reproducibility of High-Throughput Plate-Reader

476

Experiments in Synthetic Biology. ACS Synth. Biol. 2017, 6 (2), 375–380.

477

https://doi.org/10.1021/acssynbio.6b00198.

ACS Paragon Plus Environment

ACS Synthetic Biology 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

478

(47)

Page 20 of 20

Kleinstiver, B. P.; Prew, M. S.; Tsai, S. Q.; Topkar, V. V.; Nguyen, N. T.; Zheng, Z.;

479

Gonzales, A. P. W.; Li, Z.; Peterson, R. T.; Yeh, J. R. J.; et al. Engineered CRISPR-

480

Cas9 Nucleases with Altered PAM Specificities. Nature 2015, 523 (7561), 481–485.

481

https://doi.org/10.1038/nature14592.

482

(48)

Kleinstiver, B. P.; Prew, M. S.; Tsai, S. Q.; Nguyen, N. T.; Topkar, V. V.; Zheng, Z.;

483

Joung, J. K. Broadening the Targeting Range of Staphylococcus Aureus CRISPR-Cas9

484

by Modifying PAM Recognition. Nat. Biotechnol. 2015, 33 (12), 1293–1298.

485

https://doi.org/10.1038/nbt.3404.

486

(49)

Hu, J. H.; Miller, S. M.; Geurts, M. H.; Tang, W.; Chen, L.; Sun, N.; Zeina, C. M.; Gao,

487

X.; Rees, H. A.; Lin, Z.; et al. Evolved Cas9 Variants with Broad PAM Compatibility and

488

High

489

https://doi.org/10.1038/nature26155.

490

(50)

DNA

Specificity.

Nature

2018,

556

(7699),

57–63.

Hirano, H.; Gootenberg, J. S.; Horii, T.; Abudayyeh, O. O.; Kimura, M.; Hsu, P. D.;

491

Nakane, T.; Ishitani, R.; Hatada, I.; Zhang, F.; et al. Structure and Engineering of

492

Francisella

493

https://doi.org/10.1016/j.cell.2016.01.039.

494

(51)

Novicida

Cas9.

Cell

2016,

164

(5),

950–961.

Rose, J. C.; Stephany, J. J.; Valente, W. J.; Trevillian, B. M.; Dang, H. V.; Bielas, J. H.;

495

Maly, D. J.; Fowler, D. M. Rapidly Inducible Cas9 and DSB-DdPCR to Probe Editing

496

Kinetics. Nat. Methods 2017, 14 (9), 891–896. https://doi.org/10.1038/nmeth.4368.

497

(52)

Chatterjee, A.; Xiao, H.; Schultz, P. G. Evolution of Multiple, Mutually Orthogonal Prolyl-

498

TRNA Synthetase/TRNA Pairs for Unnatural Amino Acid Mutagenesis in Escherichia

499

Coli.

500

https://doi.org/10.1073/pnas.1212454109.

501

(53)

Proc.

Natl.

Acad.

Sci.

2012,

109

(37),

14841–14846.

Lee, T. S.; Krupa, R. A.; Zhang, F.; Hajimorad, M.; Holtz, W. J.; Prasad, N.; Lee, S. K.;

502

Keasling, J. D. BglBrick Vectors and Datasheets: A Synthetic Biology Platform for Gene

503

Expression. J. Biol. Eng. 2011, 5, 15–17. https://doi.org/10.1186/1754-1611-5-12.

504

(54)

Werner, S.; Engler, C.; Weber, E.; Gruetzner, R.; Marillonnet, S. Fast Track Assembly

505

of Multigene Constructs Using Golden Gate Cloning and the MoClo System. Bioeng.

506

Bugs 2012, 3 (1), 38–43. https://doi.org/10.4161/bbug.3.1.18223.

507 508

ACS Paragon Plus Environment