Nontransgenic Marker-Free Gene Disruption by an ... - ACS Publications

Subscriber access provided by - Access paid by the | UCSB Libraries

Non-transgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779 Eric Poliner, Tomomi Takeuchi, Zhi-Yan Du, Christoph Benning, and Eva Farre ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00362 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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 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

ACS Synthetic Biology

1

Non-transgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous

2

microalga, Nannochloropsis oceanica CCMP1779

3

Eric Poliner1,2, Tomomi Takeuchi2,3, Zhi-Yan Du2,3, Christoph Benning2,3,4, Eva M. Farré4*

4 5

1. Cell and Molecular Biology Program, Michigan State University, East Lansing, Michigan

6

2. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan

7

3. Biochemistry and Molecular Department, Michigan State University, East Lansing, Michigan

8

4. Plant Biology Department, Michigan State University, East Lansing, Michigan

9

*Corresponding author

10 11

Abstract

12

Utilization of microalgae has been hampered by limited tools for creating loss-of-function

13

mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic

14

resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous

15

microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid

16

metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that

17

enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces

18

cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal

19

DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked

20

sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal

21

DNA after the removal of selection pressure, resulting in marker-free non-transgenic engineered

22

lines. To test this system, we disrupted the nitrate reductase gene (NR) and subsequently

23

removed the CRISPR episome to generate non-transgenic marker-free nitrate reductase knockout

24

lines (NR-KO).

25 26 27 28

Keywords: CRISPR/Cas9, Nannochloropsis, episome, marker-free, gene targeting, algal genetic

29

engineering

30

1

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

31

Microalgae are some of the most productive biomass sources on Earth and can generate many

32

high-value bioproducts such as omega-3 fatty acids, carotenoids, and unusual polysaccharides

33

(1). One genus of microalgae that has distinguished itself for productivity and genetic

34

engineering potential is Nannochloropsis (2-4). Genetic engineering of microalgae has been

35

hampered by a limited ability to disrupt genes and by the challenges of generating transformed

36

algae that meet biocontainment requirements for open pond production (5). Recently, the use of

37

endogenous elements, marker-free strategies, and DNA free genome editing have allowed the

38

generation of modified organisms that lack antibiotic selection markers (6-8) or even any

39

heterologous DNA (9, 10). Such organisms satisfy the criteria for non-transgenic organisms,

40

making deployment of modified strains into open systems feasible.

Page 2 of 25

41 42

Gene targeting in microalgae has been performed by homologous recombination (11), TALENs

43

(12-14), and CRISPR/Cas9 (10, 15-20). Gene disruption by these methods in microalgae has

44

usually required the integration of a transgenic selection marker into the genome. A

45

CRISPR/Cas9 system for N. oceanica IMET had low efficiency (~1-4%), possibly due to low

46

targeting efficiency of the single guide RNA (sgRNA) produced from a protein-coding gene

47

promoter (18). Another CRISPR/Cas9 system for N. gaditana utilized introduction of

48

synthesized sgRNAs and required two selection markers for targeted disruption (17). Efficient

49

CRISPR/Cas9 based mutagenesis requires a sgRNA without modified ends or extraneous

50

sequences for efficient interaction with the Cas9 nuclease and DNA target. Therefore, sgRNAs

51

flanked by the hammerhead (HH) and hepatitis delta virus (HDV) self-cleaving ribozymes are

52

used to generate precise 5’ and 3’ ends (21, 22), and enable the production of sgRNAs under the

53

control of protein-coding gene promoters (RNA polymerase II class).

54 55

Episomes (extrachromosomal nuclear DNA that is usually circular) occur naturally in some red

56

algae (23), and diatoms are capable of maintaining synthetic circular episomes containing a low

57

GC (guanine cytosine) centromere-like region (24-26). In diatoms, an extrachromosomal

58

episome with a centromere and autonomous replication sequence fusion developed from

59

Saccharomyces cerevisiae (CEN/ARS) is replicated and segregated into daughter cells (24, 25).

60

Furthermore, this episomal system is able to produce proteins that can be localized throughout

61

the cell and results in more uniform transgene expression levels between independent 2


Page 3 of 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


62

transformants compared to integrated vectors, possibly by avoiding integration site-specific

63

effects (25). While circular episomes are an effective expression platform in diatoms, without

64

selection pressure, they are lost over several generations providing a way to “cure” cells of them

65

(25-27).

66 67

We developed a CRISPR/Cas9 system that can be expressed from an episome in the heterokont

68

microalgae Nannochloropsis oceanica CCMP1779. The subsequent removal of the episome after

69

the mutation was produced generates marker-free non-transgenic gene disruption mutants that

70

can be modified repeatedly.

71 72

Results/Discussion

73

The linearized pNOC-CRISPR-GFP vector, containing a Cas9-GFP expression cassette and a

74

Hygromycin B resistance marker (HygR ) (3), generated Hygromycin B-resistant transformants

75

when introduced into N. oceanica (Figure 1A). To establish a highly efficient CRISPR/Cas9

76

gene editing system in N. oceanica we confirmed the nuclear localization of a Cas9-GFP

77

nuclease with C’ and N’ terminal SV40 nuclear localization signals (NLS) by confocal

78

microscopy. Wild-type lines did not possess any GFP signal, while an untargeted GFP

79

expressing line had GFP signal throughout the cytosol with partial overlap with the DAPI stain

80

(Figure S1A-B). The Cas9-GFP signal was punctate and co-localized with a DNA stain (4’, 6-

81

Diamidino-2-Phenylindole, DAPI), demonstrating Cas9-GFP nuclear localization in N. oceanica

82

(Figure 1B, Figure S1C). To facilitate Cas9 detection in vivo, we then generated a Cas9 nuclease

83

fused to the ultra-bright NanoLuciferase reporter protein with an HA tag (Nlux-HA) in the vector

84

pNOC-CRISPR. Nlux has high luminescence activity (28) and accordingly, N. oceanica

85

transformants can be screened for the presence of the Cas9-Nlux-HA fusion in a 96-well plate

86

using a small number of cells (2). Immunoblotting confirmed the production of Cas9-Nlux-HA

87

(186.2 kDa) and Cas9-GFP proteins (191.8 kDa) (Figure 1C-D) in lines transformed with the

88

integrating empty vector constructs (iEV) pNOC-CRISPR and pNOC-CRISPR-GFP

89

respectively. These constructs lack a sgRNA sequence for a specific genomic locus.

90 91

The pNOC-CRISPR vector contains the Cas9-Nlux-HA and a gRNA scaffold with a 3’ fusion to

92

the HDV ribozyme (21, 22) under the control of the recently characterized endogenous 3



93

ribosomal subunit bidirectional promoter (Ribi, located between NannoCCMP1779_9669 and

94

NannoCCMP1779_9668) (2). To facilitate cleavage precisely at the 5’ end of as gRNA

95

sequence, a hammerhead ribozyme (HH) specific to the guide sequence is introduced during

96

sgRNA generation (Figure S2). Strategies for cloning sgRNAs are described in the Methods

97

section.

Page 4 of 25

98 99

We next designed an episome-based CRISPR/Cas9 system, by incorporating the CEN/ARS

100

region from S. cerevisiae into the pNOC-CRISPR-Nlux vector, generating the vector pNOC-

101

ARS-CRISPR (Figure 2A). To test this system, we targeted the nitrate reductase gene (NR) for

102

the known phenotype of NR disruption lines (NR-KO) (Figure 2A) (6, 29, 30). Nannochloropsis

103

NR-KO lines can grow on ammonium (NH4) but are not able to sustain growth when provided

104

with only nitrate (NO3) as a nitrogen source (11, 18). Two sgRNAs flanked by self-cleaving

105

ribozymes (sgNR1 and sgNR2) were used to independently target two sites at the 5’ end of the

106

NR gene. We introduced the pNOC-ARS-CRISPR-sgNR1 and pNOC-ARS-CRISPR-sgNR2

107

episomes into N. oceanica and generated the NR1-KO and NR2-KO lines respectively.

108

Introduction of the circular episomal CRISPR plasmids into N. oceanica resulted in Hygromycin

109

B-resistant lines, which were screened for Cas9-Nlux-HA signal (Figure S3A-B).

110 111

Lines displaying luminescence were examined for mutations in the NR gene by PCR

112

amplification of the genomic locus and Sanger sequencing (Figure S3). We found cumulatively

113

that 47% of lines with Nlux signal had frame-shift mutations and 13% had in-frame mutations,

114

while 13% had no mutation and 27% resulted in low-quality sequencing outcomes (Figure S3C).

115

The two sgRNAs had different efficiencies with sgNR1 (in NR1-KO lines) having a high

116

mutation efficiency with 9/10 screened colonies containing a mutation and no wild-type

117

sequences recovered, while sgNR2 (in NR2-KO lines) had a lower mutation efficiency with 9/20

118

screened lines possessing a mutation and 4/20 lines lacking a mutation (Figure S3C). The low-

119

quality sequencing reactions often had good quality chromatographs up to the target site and then

120

became unreadable. This observation suggests that a heterogeneous population of mutants was

121

recovered from single colonies, possibly due to mutagenesis occurring after plating. It is likely

122

that pure genotypic lines could be obtained from these populations by streaking to single

123

colonies. 4


Page 5 of 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


124 125

We selected N. oceanica mutants with small deletions (1D, 1H), a larger (47 bp) deletion (4G),

126

and a small insertion (B12) as frame-shifted null mutants (NR-KO lines), and the B7 line with a

127

3 bp deletion as an in-frame mutant (NR-IF) for further analyses (Figure 2B, Figure 3D-E).

128

Immunoblotting detected the Cas9-Nlux-HA protein in all the lines indicating that proteins can

129

be produced from an episomal DNA in N. oceanica (Figure 2C). To test for the loss of NR

130

activity, cell growth was analyzed in liquid cultures after transfer from NH4 to NO3 containing

131

medium. The frame-shifted NR-KO mutants could not grow, while the wild-type (WT) lines, the

132

iEV lines, and the in-frame mutant B7 grew in NO3-containing liquid medium (Figure 2D). This

133

indicates frame-shifts introduced into the coding sequence of targeted genes ablate the function

134

of the resulting protein, while in-frame mutations may still produce functional proteins.

135 136

To determine if the episomes were maintained as circular DNA, we conducted an episome rescue

137

experiment by transforming Escherichia coli with DNA isolated from N. oceanica episome-

138

carrying lines (NR-KOs). WT and iEV lines were used as negative controls. E. coli

139

transformants were only obtained from untreated DNA isolated from NR-KO lines, but not from

140

untreated DNA of iEV lines, or the WT strain (Figure 2E). To further confirm that the plasmids

141

rescued from N. oceanica NR-KO lines were circular, an equal amount of DNA from the NR-

142

KO lines was subjected to endonuclease ClaI restriction digest and/or treated with Exonuclease

143

V, which acts on free DNA ends(25). Due to the ClaI site in the pNOC-ARS-CRISPR plasmid,

144

ClaI treatment of DNA isolated from NR-KO lines strongly decreased the number of E. coli

145

transformants (Figure 2E). Treatment with Exonuclease V resulted in a small reduction of E. coli

146

transfomants compared to mock treatments, while a combined endonuclease and exonuclease

147

treatment resulted in very few colonies being recovered (Figure 2E). Restriction fragment

148

analysis of plasmids recovered from the episome rescue indicated that the episomes were

149

faithfully maintained in N. oceanica (Figure S4). Restriction enzyme recognition sites by the

150

CEN/ARS region (AgeI), in the Cas9 and HygR genes (EcoRI), and backbone (NotI), indicated

151

that the episome had no apparent insertions or deletions (Figure S4A-B). Sequencing of the

152

recovered episomes further confirmed their authenticity (Figure S4C-D).

153

5



Page 6 of 25

154

We performed southern blots to further characterize the CRISPR episomal lines (Figure S5). As

155

a control, we used iEV lines containing the genomic integrated pNOC-CRISPR vector. DNA

156

was isolated from NR-KO, iEV and WT cells and was then digested with SacI and hybridized

157

with either a HygR or an AmpR gene probe (Figure S5A). In all the NR-KO lines tested, both

158

probes generated one fragment of ~13,000 bp that matched the episome CRISPR vector size

159

(Figure S5B). However, the iEV lines generated different size fragments when using the HygR

160

probe and only one line had a detectable fragment when the AmpR probe was used (Figure S5B).

161

The AmpR probe hybridizes close to the AseI cut site used for linearizing the pNOC-CRISPR

162

vector before transformation to generate the iEV lines. Therefore, the absence of a band in

163

AmpR probed iEV DNA indicates that the AmpR was partially lost during integration or that the

164

fragment is too small to be detected in the current experiment. None of the WT lines had a

165

detectable signal in these assays. This experiment further confirmed the presence of a circular

166

DNA molecule in the CRISPR episomal lines.

167 168

After generating the NR-KO lines we sought to remove the episome to form marker-less non-

169

transgenic mutants. Nlux signal is a convenient measure for Cas9 presence in the transformed

170

lines and to monitor for the loss of the episomes. Four NR-KO lines were grown for 10 days

171

without selection in liquid medium and plated on solid medium to generate independent colonies.

172

The colonies were screened for Nlux luminescence, followed by a PCR test for the presence of

173

the episome and a control genomic locus (Figure S6A). After growth in non-selective medium,

174

the NR-KO lines had various levels of Nlux signal and 25-75% of the lines had low

175

luminescence signal (Figure S6B). The rate of successful inheritance of an episome during each

176

cell division (segregation efficiency) is ascertained by removing selection for a known number of

177

cell divisions and determining the percentage of episome carrying individuals (25, 31). In order

178

to determine the number of generations that occurred during the episome curing procedure, the

179

doubling time (1.023 days) of N. oceanica grown in NH4 media was measured (Figure S7). After

180

~30 generations, the segregation efficiency of the pNOC-CRISPR constructs was 96-99% based

181

on the percentage of recovered colonies that maintained luminescence signal (Figure S6B).

182 183

To confirm that these cured low Nlux luminescent lines had lost the episome, we conducted PCR

184

for the Cas9 gene contained in the episome, and the NR gene as a genomic positive control 6


Page 7 of 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


185

(Figure 3A-B). PCR detected the NR gene in all lines, while the Cas9 coding sequence was only

186

present in the episomal lines but not in the cured lines (Figure 3B). Furthermore, growth tests

187

were used to demonstrate the different phenotypes of the generated lines. First, the cured lines

188

that had lost the episome were unable to grow on Hygromycin B-containing medium, while the

189

episome-carrying parental lines were Hygromycin B resistant (Figure 3C). Second, growth on

190

solid medium containing either NO3 or NH4 revealed a chlorotic phenotype of the episome-

191

carrying and cured NR-KO lines only when grown on NO3 (Figure 3C) (11).

192 193

We have developed a system to produce marker-less non-transgenic gene knockout strains that

194

not only theoretically allows for the generation of lines with multiple modifications but also

195

facilitates the transfer of strains developed in the lab to the field. Our CRISPR/Cas9 components

196

have been optimized for efficient detection, with a Cas9-Nlux-HA reporter fusion that has high

197

signal to noise ratio and can be screened in a 96-well plate format soon after transformant

198

isolation. Furthermore, the Cas9-Nlux-HA and sgRNA are coregulated by a bidirectional

199

promoter, thus linking Cas9 production to sgRNA expression (Figure 1A) (2). The pNOC-

200

CRISPR vector series contains unique restriction sites between the elements making it an ideal

201

platform for further development of CRISPR/Cas9 as a system for transcriptional

202

reprogramming and other DNA targeting techniques.

203 204

The use of self-cleaving ribozymes facilitates the flexible expression sgRNAs. The ribozyme

205

flanked sgRNAs can be expressed from different types of promoters, in our case a bidirectional

206

promoter, but also possibly from conditional promoters. The differing targeting efficiencies of

207

the sgRNAs tested in this study demonstrate that sgRNA design plays an important role in Cas9

208

gene disruption rates (Figure S3C), and indicates that the development of sgRNA design tools

209

(32, 33) for this genus is a route for further optimization of CRISPR/Cas9 in Nannochloropsis.

210

Our final episomal vector, pNOC-ARS-CRISPR-v2, contains a multi-cloning site with a pair of

211

BspQI type IIS restriction sites for scarless insertion of a guide sequence, for one-step cloning

212

(Figure S8A-B). Self-cleaving ribozyme sequences for expression of the sgRNA could be used

213

for the production of multiple sgRNAs from a single transcript (22). Production of multiple

214

sgRNAs can be used to target multiple genes for simultaneous disruption, used in pairs with

7



215

nickase Cas9 variants for enhanced specificity, or for deletion of a region between two targeted

216

sites on a chromosome.

Page 8 of 25

217 218

Transgenic expression in algae is most often performed by integrating a DNA construct into the

219

genome. However, this can result in site-specific effects, or the disruption of genes at the

220

insertion site. Therefore, episomes are an emerging expression platform in microalgae that

221

avoids these issues. In diatoms, effective centromere sequences have the simple requirements of

222

a GC content 500 bp, with long stretches of A-T sequences (24-26). These

223

centromere-like regions interact with the centromeric histone protein (CENH3) likely facilitating

224

segregation during cell division (24). The segregation efficiency of the pNOC-CRISPR system

225

was 96-99% (Figure S6B) which is comparable to the 97% segregation efficiency reported for

226

episomal constructs developed for diatoms (25). However, the molecular processes for

227

replication of synthetic episomes in algae are yet to be determined. The discovery of the

228

relatively simple sequence requirements for centromeres in diatoms and the observation that the

229

yeast CEN/ARS region is effective in diatoms and Nannochloropsis species suggest that

230

heterokont algae have the potential to maintain newly acquired DNA, facilitating gene

231

acquisition.

232 233

While first-generation synthetic episomes can robustly express transgenes, they are gradually lost

234

from the population when selection pressure is not applied. These characteristics are ideal for

235

transient CRISPR/Cas9 gene disruption, making it easy to screen transformants for mutations in

236

the target site, followed by curing of the episome (8, 9). The cured mutants, thus contain a scar in

237

the target site but lack an antibiotic resistance gene or any other transgenic elements, and can be

238

used for subsequent modifications and do not need further biocontainment strategies. This

239

technique will aid in the development of chassis strains for biotechnology, and makes marker-

240

free genomic integration or endogenous locus tagging of expression constructs foreseeable.

241 242

To make the generated non-transgenic NR-KO mutant available for wide use, the NR1-KO 4G-

243

5A strain is deposited with NCMA (Table S1). To make the high-efficiency, CRISPR/Cas9 dual

244

component pNOC-CRISPR vectors widely available, the plasmids developed in this study are

245

deposited with Addgene (Table S1). 8


Page 9 of 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


246 247

Methods

248

Strains and growth conditions

249

N. oceanica CCMP1779 was used in all experiments. Cells were grown in F/2 medium under

250

constant 100 µmol m-2 s-1 light at 22º C, on a shaker set at 120 rpm. F/2 medium with 2.5 mM

251

KNO3 or 2.5 mM NaNH4 was used. Hygromycin B (Sigma-Aldrich) was utilized at a

252

concentration of 100 µg/ml. Cell concentrations were measured with a Z2 Coulter Counter

253

(Beckman Coulter) with a range of 1.8-3.6 µM. To determine whether growth could be sustained

254

on nitrate, N. oceanica strains were inoculated to 5x106 cells/ml in F/2 with 2.5 mM KNO3 and

255

cell density measured every 24 hours. For assessment of antibiotic resistance, growth after

256

plating 30,000 cells on solid medium was monitored for 1 month.

257 258

CRISPR plasmid construction

259

The details of the construction of the plasmids produced in this study is contained in Data S1.

260 261

N. oceanica transformation

262

Integrating vectors were digested with AseI and concentrated by ethanol precipitation.

263

Transformations were conducted as previously described (2), with 3 µg of DNA and 30 µg of

264

blocking DNA (Ultrapure salmon sperm DNA - Invitrogen). Transformants were plated using

265

the top agar method on 100 µg/ml Hygromycin B and grown under 100 µmol m-2 s-1 for 3 weeks.

266

Individual lines were transferred to 500 µl of F/2 with 100 µg/ml Hygromycin B (GoldBio) in a

267

96 deep well plate (Evergreen Biotech). Cultures were then maintained on solid plates.

268 269

NanoLuciferase luminescence assays

270

For Nlux activity screening, 100 µl of cell culture was transferred to a luminescence plate, and

271

100 µl of F/2 containing Nanoglo substrate (Promega) at a 10,000X dilution. For normalized

272

luminescence measurements, 1.5 million cells per well were transferred to the 96-well

273

luminometer plate, the volume was adjusted to 100 µl with F/2 medium, and 100 µl of F/2

274

containing Nanoglo substrate (Promega) at a 10,000X dilution was added. Luminescence was

275

measured after 180 sec delay with a 0.3 sec exposure using a Centro XS3 LB960 (Berthold).

276

9



Page 10 of 25

277

N. oceanica colony PCR

278

A 1X Q5 buffered solution (NEB) with 1 µl of N. oceanica culture per 10 µl of required sample

279

was boiled (100º C) for 10 min. A 10 µl PCR mastermix was added to the 10 µl of sample and

280

PCR conducted according to the manufacturer’s suggestions (NEB). The primers NR F+ and NR

281

R- were used to amplify the NR gene (Table S2).

282 283

Episomal DNA isolation from N. oceanica

284

For DNA isolation 10 ml of culture at mid-log phase (~ 3x107 cells ml-1) was collected and

285

frozen. The protocol of Karas et al. (25) was utilized with some modifications. Briefly, for each

286

reaction 235 µl Qiagen P1 solution was combined with 5 µl lysozyme (25 mg/ml) (Sigma-

287

Aldrich), 2.5 µl macerozyme (100 mg/ml)(Yakult Pharmaceuticals), 2.5 µl zymolyase (10

288

mg/ml) (Zymo Research), and 5 µl cellulase (100mg/ml)(Yakult Pharmaceuticals) and used to

289

resuspend the cell pellets. The resuspended cell culture was incubated at 37º C for 30 min. After

290

addition of the P2 solution, and S3 solution (Qiagen), the cell debris was pelleted at 14000 x G

291

for 10 min. Supernatant was aspirated and combined with an equal volume of isopropanol, mixed

292

and centrifuged at 14,000 x G at 4º C for 10 min. After decanting the pellet was washed with 750

293

µl 70% ethanol, centrifuged, decanted, and dried. The isolated DNA was resuspended in Tris-

294

EDTA buffer (TE) and re-isolated by phenol:chloroform extraction. The final DNA was

295

resuspended in 40 µl TE, and the concentration determined using a NanoDrop Lite (Thermo

296

Scientific).

297 298

Episome rescue

299

Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli

300

transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 µg of DNA

301

was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 µl

302

reaction for 1 hour at 37º C. Reactions were heat inactivated at 75º C for 30 min. An equal

303

amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples

304

were used to transform E. coli (DH5α high-efficiency efficiency, NEB). Resulting colonies were

305

counted and random colonies selected for plasmid isolation. Resulting plasmids and control

306

pNOC-ARS-CRISPR-sgNR2 DNA, were digested with AgeI, NotI, and EcorI (NEB) according

307

to manufacturer’s instructions (NEB), and separated on a 0.9% agarose gel. 10


Page 11 of 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


308 309

Episome curing

310

Episome containing NR-KO lines were grown in liquid 2.5 mM NaNH4 F/2 without Hygromycin

311

B for 10 days. Cultures were diluted to 3,000 cells per ml, and 3,000 cells were plated on NaNH4

312

F/2. Single colonies developed over 3 weeks, and 48 colonies were isolated and grown in NaNH4

313

F/2. After 1 week, the Nlux luminescence was measured and low Nlux signal lines were further

314

screened by colony PCR. Primers used were NR F+, NR R-, and epi Cas9 F+and epi Cas9 R-

315

(Table S2).

316 317

Segregation efficiency

318

A growth curve was conducted using an inoculum of ~5x106 cells/ml in F/2 with 2.5 mM NaNH4

319

and cell density was measured every 24 hours. The doubling time was determined using a best-fit

320

exponential curve and the number of generations deduced by dividing the duration of the

321

experiment by the doubling time. The segregation efficiency was determined according to

322

Iwanaga et al. (25, 31) and using the percentage of cells that maintained luminescence in the

323

curing screen.

324 325

Immunoblotting

326

Immunoblotting was conducted as described previously (2). Protein extract (50 µg) was

327

separated on 8-10% SDS-polyacrylamide gels. After transfer to PVDF membranes Cas9-GFP

328

was detected with α-GFP (Abcam ab5450) 1:1,000 in TBST with 5% BSA followed by donkey

329

α-goat-HRP (Santa Cruz sc-2020) 1:10,000 in TBST with 5% milk, and Cas9-Nlux-HA with α-

330

HA-HRP (Roche 3F10) 1:1,000 in TBST with 5% milk. Detection was conducted by

331

chemilumiscence with Femto substrate (ThermoFisher). After detection, total protein was stained

332

with Direct Blue 71 (34).

333 334

Confocal microscopy

335

Confocal microscopy was performed using an inverted Olympus FluoView1000 confocal laser

336

scanning microscope (Olympus Corporation, USA). Cells grown in F/2 medium were fixed in

337

70% ethanol overnight at 4°C. Cell pellets were collected and stained with 4’, 6-Diamidino-2-

338

Phenylindole (DAPI, 358/461 - Life Technologies) with a final concentration of 0.2 µg/µl in 11



Page 12 of 25

339

PBS for 2 hours at 4°C. Cells were washed with PBS and observed using a 100x UPlanSApo oil

340

objective (N.A. 1.4). DAPI fluorochromes were excited using a 405 nm blue diode laser, and the

341

emission signals were filtered using the BA430-470 band pass filter. Cas9-GFP was excited

342

using an argon 488 nm laser, and the emission signals from GFP were filtered using a BA500-

343

530 band pass filter. Post-imaging analyses were performed using Olympus FluoView1000

344

software.

345 346

Southern-blot analysis

347

We used a procedure described previously (3). Briefly, N. oceanica DNA was isolated by CTAB

348

and 20 µg was digested with SacI and was separated on an agarose gel (0.9%, 70 Volts

349

overnight), followed by blotting to a Hybond Nylon membrane overnight (GE Health Care).

350

Subsequent hybridization and detection was performed with a DIG labeling and detection kit

351

according to the manufacturer’s instructions (Roche Applied Sciences). Two probes were

352

amplified by PCR, one for the HygR gene (with the primers HygR probe F+ and HygR probe R-)

353

and one for the AmpR gene (AmpR probe F+ and AmpR probe R-), and were used for

354

hybridization in PefectHyb Plus hybridization buffer (Sigma-Aldrich).

355 356

Figures and captions

357 358

Figure 1. A one-vector CRISPR system for gene disruption in N. oceanica. (A) The pNOC-

359

CRISPR vector series includes a Hygromycin B resistance cassette (HygC, green). A

360

bidirectional promoter (Ribi) drives the transcription of the Cas9-reporter fusion and gRNA

361

scaffold (scaffold, blue), along with the LDSP and CS terminators (LDSP-T, CS-T) respectively.

12


Page 13 of 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


362

Cas9 is fused to either the GFP or Nlux (with HA tag) reporters by a 3x glycine-serine linker

363

(GSGSGS) and contains SV40 nuclear localization signals on the N’ and C’ termini (NLS). The

364

gRNA scaffold and the 3’ self-cleaving HDV ribozyme is integrated into the vector. The 5’

365

hammerhead ribozyme (HH) specific for each guide sequence (GS, orange) is fused to the gRNA

366

scaffold (scaffold) to form a sgRNA. Ribozymes are highlighted in yellow. Unique restriction

367

sites are shown with an upwards line and the name in italics. (B) Confocal analysis of DAPI

368

nuclear staining, Cas9-GFP signal, and merged brightfield, DAPI and GFP signal in N. oceanica

369

cells. Scale bar of 2 µM. (C) Immunoblotting with an α-GFP antibody detected the Cas9-GFP

370

produced in N. oceanica transformed with pNOC-CRISPR-GFP. A N. oceanica line producing a

371

delta-5 fatty acid desaturase (~75 kDa) fused with CFP was used as a GFP positive control (+).

372

(D) Immunoblotting with an α-HA antibody detected the appropriately sized Cas9-Nlux-HA in

373

N. oceanica transformed with pNOC-CRISPR. Wild-type N. oceanica was included as a

374

negative control (WT). For (C) and (D) numbers on the left of immunoblots indicate size

375

markers (KDa).

376 377

Figure 2. Development of an episomal CRISPR system. (A) The S. cerevisiae CEN/ARS6

378

region (ARS, red) was included in the pNOC-ARS-CRISPR construct for episomal maintenance.

379

Guide sequences(orange) for nitrate reductase targets, with a 5'hammerhead ribozyme (HH),were 13



Page 14 of 25

380

fused to the gRNA scaffold to form the NR sgRNAs (sgNR). Ribozymes are highlighted in

381

yellow. The sgNR1 and sgNR2were added to pNOC-ARS-CRISPR to form pNOC-ARS-

382

CRISPR-sgNR1 and pNOC-ARS-CRISPR-sgNR2, respectively. N. oceanica is transformed with

383

circular episomal CRISPR constructs. (B) Mutations in the two target sites in the NR genomic

384

locus (Target 1 and Target 2) of N. oceanica transformed with the respective pNOC-ARS-

385

CRISPR-sgNR construct. Mutant lines are identified by 96-well plate location (Figure S3).

386

Deleted nucleotides are represented with dashes and inserted nucleotides are shown in bold.

387

Protospacer adjacent motifs (PAM sites) are underlined. (C) Immunoblot using an α-HA

388

antibody of N. oceanicaNR1-KO and NR2-KO lines producing Cas9-Nlux-HA from the

389

CRISPR episome. Numbers on the left indicate size markers (KDa). (D) Growth curves after

390

transfer from NH4 to NO3 containing medium of NR-KO frame-shifted lines (1D, 1H, 4G, B12)

391

and NR2-IF B7 in-frame line, empty vector integrated CRISPR control lines (iEV), and wildtype

392

(WT). (E) Episome rescue by E. coli transformation using equal quantities of DNA isolated from

393

episomal (NR-KO) lines, integrated empty-vector (iEV), and wild-type (WT) N. oceanica lines.

394

Values are the average colonies generated ± SE (NR-KOs n = 3 independent lines, WT n = 2

395

biological replicates, and iEV n = 2 independent lines). Equal quantities of DNA from NR-KO

396

lines after treatment were used for E. coli transformation, and the resulting colonies counted (n =

397

3 independent lines). Exonuclease V (ExoV), ClaI endonuclease (ClaI), and ClaI endonuclease

398

with Exonuclease V (ClaI+ExoV).

399 400

Figure 3. Generation of marker-free non-transgenic mutants by episomal removal (curing). (A)

401

Luminescence from equal number of cells of wild-type (WT), and NR-KO lines either containing 14


Page 15 of 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


402

the episome or cured of the episome. (B) PCR for detection of a positive control NR genomic

403

locus and the Cas9 regions on the episome conducted on the same DNA extract obtained from

404

WT and, NR-KO episomal and cured lines. (C) Plating of an equal number of cells of WT, NR-

405

KO and NR-KO cured lines on NH4, NO3, and NH4 with Hygromycin B on F/2 solid medium

406

after 1 month of growth.

407 408

Supporting Information

409

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

410 411

Figure S1 showing confocal imaging of wildtype, untargeted GFP expressing, andCas9-GFP

412

expressing N. oceanica cells with the nucleus stained by DAPI; Figure S2 showing cloning

413

strategies for the generation of a ribozyme flanked sgRNA; Figure S3 showing identification of

414

NR knockout mutants by CRISPR/Cas9; Figure S4 showing further verification of rescued

415

episomes; Figure S5 showing southern blot analysis of the episomal and integrated empty-vector

416

CRISPR mutants; Figure S6 showing the selection of cured mutant lines; Figure S7 showing a

417

growth curve in ammonium containing medium;Figure S8 showing a one-vector CRISPR system

418

for scarless cloning of targets; Data S1 contains the description of vector construction and the

419

sequences of the vectors generated in this study (.txt); Table S1 showing the list of constructs

420

generated in this study; Table S2 showing the primers used in the study.

421 422

Abbreviations: sgRNA – single-guide RNA, CRISPR – Clustered Regularly Interspaced Short

423

Palindromic Repeats, single guide RNA, KO – knockout, HDV - hepatitis delta virus ribozyme,

424

HH - hammerhead ribozyme, CEN/ARS – Saccharomyces cerevisiae centromere and

425

autonomous replication sequence fusion, NR – nitrate reductase, HR – homologous

426

recombination, GFP – Green Fluorescent Protein, Nlux – NanoLuciferase, PCR – polymerase

427

chain reaction, DAPI - 4’, 6-Diamidino-2-Phenylindole, CLSM – confocal laser scanning

428

microscopy, NLS – nuclear localization signal

429 430

AUTHOR INFORMATION

431

Corresponding Author:

432

Eva M. Farré ([email protected]) 15



433

Page 16 of 25

Phone: +1-517-353-5215

434 435

ORCID

436

Eric Poliner: 0000-0002-9740-9034

437

Zhi-Yan Du: 0000-0001-7646-2429

438

Christoph Benning: 0000-0001-8585-3667

439

Eva M. Farré: 0000-0003-1566-7572

440 441

Author contributions

442

EP conceived the study. EP, TT and ZYD carried out the experiments. EF and CB were involved

443

in planning and supervising the work. EP and EF wrote the manuscript with input from CB. All

444

authors reviewed the final manuscript.

445 446

Note

447

The authors report no financial conflicts of interest.

448 449

Acknowledgements:

450

We thank Bill Henry for his analysis of the N. oceanica U6 region and Kathryn Meeks for the

451

gift of the hCas9 plasmid. This work was supported by a National Science Foundation grant

452

(IOS-1354721) to EF. T.T. was partly supported by the Plant Biotechnology for Health and

453

Sustainability Training Program at MSU (NIH T32 GM110523). In addition, parts of this work

454

were supported by the Division of Chemical Sciences, Geosciences and Biosciences, Office of

455

Basic Energy Sciences of the United States Department of Energy (DE-FG02-91ER20021) and

456

MSU-AgBioResearch.

457 458 459

References

460

1.

Chew, K. W., Yap, J. Y., Show, P. L., Suan, N. H., Juan, J. C., Ling, T. C., Lee, D.-J.,

461

and Chang, J.-S. (2017) Microalgae biorefinery: High value products perspectives,

462

Bioresource Technology 229, 53-62 DOI: 10.1016/j.biortech.2017.01.006

16


Page 17 of 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

463


2.

Poliner, E., Pulman, J. A., Zienkiewicz, K., Childs, K., Benning, C., and Farré, E. M.

464

(2017) A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and

465

genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain

466

polyunsaturated fatty acid production, Plant Biotechnology Journal DOI:

467

10.1111/pbi.12772

468

3.

Vieler, A., Wu, G., Tsai, C.-H., Bullard, B., Cornish, A. J., Harvey, C., Reca, I.-B.,

469

Thornburg, C., Achawanantakun, R., Buehl, C. J., Campbell, M. S., Cavalier, D., Childs,

470

K. L., Clark, T. J., Deshpande, R., Erickson, E., Armenia Ferguson, A., Handee, W.,

471

Kong, Q., Li, X., Liu, B., Lundback, S., Peng, C., Roston, R. L., Sanjaya, Simpson, J. P.,

472

TerBush, A., Warakanont, J., Zäuner, S., Farre, E. M., Hegg, E. L., Jiang, N., Kuo, M.-

473

H., Lu, Y., Niyogi, K. K., Ohlrogge, J., Osteryoung, K. W., Shachar-Hill, Y., Sears, B.

474

B., Sun, Y., Takahashi, H., Yandell, M., Shiu, S.-H., and Benning, C. (2012) Genome,

475

Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous

476

Alga Nannochloropsis oceanica CCMP1779, PLoS Genet. 8, e1003064 DOI:

477

10.1371/journal.pgen.1003064

478

4.

Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., and

479

Tredici, M. R. (2009) Microalgae for oil: Strain selection, induction of lipid synthesis and

480

outdoor mass cultivation in a low-cost photobioreactor, Biotechnology and

481

Bioengineering 102, 100-112 DOI: 10.1002/bit.22033

482

5.

9 DOI:

483 484

Kumar, S. (2015) GM Algae for Biofuel Production: Biosafety and Risk Assessment, Vol.

6.

Kindle, K. L., Schnell, R. A., Fernández, E., and Lefebvre, P. A. (1989) Stable nuclear

485

transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase,

486

J. Cell Biol. 109, 2589-2601 DOI:

487

7.

Cheah, Y. E., Albers, S. C., and Peebles, C. A. M. (2013) A novel counter-selection

488

method for markerless genetic modification in Synechocystis sp. PCC 6803,

489

Biotechnology Progress 29, 23-30 DOI: 10.1002/btpr.1661

490

8.

Lu, J., Tong, Y., Pan, J., Yang, Y., Liu, Q., Tan, X., Zhao, S., Qin, L., and Chen, X.

491

(2016) A redesigned CRISPR/Cas9 system for marker-free genome editing in

492

Plasmodium falciparum, Parasites & Vectors 9 DOI: 10.1186/s13071-016-1487-4

17



493

9.

Li, L., Gao, F., and Wu, S. (2016) An episomal CRISPR/Cas9 system to derive vector-

494

free gene modified mammalian cells, Protein & Cell 7, 689-691 DOI: 10.1007/s13238-

495

016-0299-9

496

10.

Baek, K., Kim, D. H., Jeong, J., Sim, S. J., Melis, A., Kim, J.-S., Jin, E., and Bae, S.

497

(2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9

498

ribonucleoproteins, Scientific Reports 6, 30620 DOI: 10.1038/srep30620

499

11.

Page 18 of 25

Kilian, O., Benemann, C. S. E., Niyogi, K. K., and Vick, B. (2011) High-efficiency

500

homologous recombination in the oil-producing alga Nannochloropsis sp, Proceedings of

501

the National Academy of Sciences 108, 21265-21269 DOI: 10.1073/pnas.1105861108

502

12.

Daboussi, F., Leduc, S., Maréchal, A., Dubois, G., Guyot, V., Perez-Michaut, C., Amato,

503

A., Falciatore, A., Juillerat, A., Beurdeley, M., Voytas, D. F., Cavarec, L., and

504

Duchateau, P. (2014) Genome engineering empowers the diatom Phaeodactylum

505

tricornutum for biotechnology, Nature Communications 5 DOI: 10.1038/ncomms4831

506

13.

Diner, R. E., Schwenck, S. M., McCrow, J. P., Zheng, H., and Allen, A. E. (2016)

507

Genetic Manipulation of Competition for Nitrate between Heterotrophic Bacteria and

508

Diatoms, Frontiers in Microbiology 7 DOI: 10.3389/fmicb.2016.00880

509

14.

Weyman, P. D., Beeri, K., Lefebvre, S. C., Rivera, J., McCarthy, J. K., Heuberger, A. L.,

510

Peers, G., Allen, A. E., and Dupont, C. L. (2015) Inactivation of Phaeodactylum

511

tricornutum urease gene using transcription activator-like effector nuclease-based

512

targeted mutagenesis, Plant biotechnology journal 13, 460-470 DOI: 10.1111/pbi.12254

513

15.

Jiang, W., Brueggeman, A. J., Horken, K. M., Plucinak, T. M., and Weeks, D. P. (2014)

514

Successful Transient Expression of Cas9 and Single Guide RNA Genes in

515

Chlamydomonas reinhardtii, Eukaryotic Cell 13, 1465-1469 DOI: 10.1128/ec.00213-14

516

16.

Shin, S.-E., Lim, J.-M., Koh, H. G., Kim, E. K., Kang, N. K., Jeon, S., Kwon, S., Shin,

517

W.-S., Lee, B., Hwangbo, K., Kim, J., Ye, S. H., Yun, J.-Y., Seo, H., Oh, H.-M., Kim,

518

K.-J., Kim, J.-S., Jeong, W.-J., Chang, Y. K., and Jeong, B.-r. (2016) CRISPR/Cas9-

519

induced knockout and knock-in mutations in Chlamydomonas reinhardtii, Scientific

520

Reports 6, 27810 DOI: 10.1038/srep27810

521

17.

Ajjawi, I., Verruto, J., Aqui, M., Soriaga, L. B., Coppersmith, J., Kwok, K., Peach, L.,

522

Orchard, E., Kalb, R., Xu, W., Carlson, T. J., Francis, K., Konigsfeld, K., Bartalis, J.,

523

Schultz, A., Lambert, W., Schwartz, A. S., Brown, R., and Moellering, E. R. (2017) Lipid 18


Page 19 of 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


524

production in Nannochloropsis gaditana is doubled by decreasing expression of a single

525

transcriptional regulator, Nature Biotechnology 35, 647-652 DOI: 10.1038/nbt.3865

526

18.

Wang, Q., Lu, Y., Xin, Y., Wei, L., Huang, S., and Xu, J. (2016) Genome editing of

527

model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9, Plant J 88, 1071-

528

1081 DOI: 10.1111/tpj.13307

529

19.

Hopes, A., Nekrasov, V., Kamoun, S., and Mock, T. (2016) Editing of the urease gene by

530

CRISPR-Cas in the diatom Thalassiosira pseudonana, Plant Methods 12 DOI:

531

10.1186/s13007-016-0148-0

532

20.

Nymark, M., Sharma, A. K., Sparstad, T., Bones, A. M., and Winge, P. (2016) A

533

CRISPR/Cas9 system adapted for gene editing in marine algae, Scientific Reports 6,

534

24951 DOI: 10.1038/srep24951

535

21.

Leishmania donovani, mBio 6, e00861-00815 DOI: 10.1128/mBio.00861-15

536 537

Zhang, W.-W., and Matlashewski, G. (2015) CRISPR-Cas9-Mediated Genome Editing in

22.

Weninger, A., Hatzl, A.-M., Schmid, C., Vogl, T., and Glieder, A. (2016) Combinatorial

538

optimization of CRISPR/Cas9 expression enables precision genome engineering in the

539

methylotrophic yeast Pichia pastoris, Journal of Biotechnology 235, 139-149 DOI:

540

10.1016/j.jbiotec.2016.03.027

541

23.

Moon, D. A., and Goff, L. J. (1997) Molecular characterization of two large DNA

542

plasmids in the red alga Porphyra pulchra, Curr. Genet. 32, 132-138 DOI:

543

10.1007/s002940050257

544

24.

Diner, R. E., Noddings, C. M., Lian, N. C., Kang, A. K., McQuaid, J. B., Jablanovic, J.,

545

Espinoza, J. L., Nguyen, N. A., Anzelmatti, M. A., Jansson, J., Bielinski, V. A., Karas, B.

546

J., Dupont, C. L., Allen, A. E., and Weyman, P. D. (2017) Diatom centromeres suggest a

547

mechanism for nuclear DNA acquisition, Proceedings of the National Academy of

548

Sciences 114, E6015-E6024 DOI: 10.1073/pnas.1700764114

549

25.

Karas, B. J., Diner, R. E., Lefebvre, S. C., McQuaid, J., Phillips, A. P. R., Noddings, C.

550

M., Brunson, J. K., Valas, R. E., Deerinck, T. J., Jablanovic, J., Gillard, J. T. F., Beeri,

551

K., Ellisman, M. H., Glass, J. I., Hutchison Iii, C. A., Smith, H. O., Venter, J. C., Allen,

552

A. E., Dupont, C. L., and Weyman, P. D. (2015) Designer diatom episomes delivered by

553

bacterial conjugation, Nature Communications 6, 6925 DOI: 10.1038/ncomms7925

19



554

26.

Diner, R. E., Bielinski, V. A., Dupont, C. L., Allen, A. E., and Weyman, P. D. (2016)

555

Refinement of the Diatom Episome Maintenance Sequence and Improvement of

556

Conjugation-Based DNA Delivery Methods, Frontiers in bioengineering and

557

biotechnology 4, 65 DOI: 10.3389/fbioe.2016.00065

558

27.

Page 20 of 25

Slattery, S. S., Diamond, A., Wang, H., Therrien, J. A., Lant, J. T., Jazey, T., Lee, K.,

559

Klassen, Z., Desgagne-Penix, I., Karas, B. J., and Edgell, D. R. (2018) An Expanded

560

Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance

561

of Synthetic Pathways in Phaeodactylum tricornutum, ACS Synth Biol DOI:

562

10.1021/acssynbio.7b00191

563

28.

Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., Otto,

564

P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A., Eggers,

565

C. T., Slater, M. R., Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P., and

566

Wood, K. V. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a

567

novel imidazopyrazinone substrate, ACS chemical biology 7, 1848-1857 DOI:

568

10.1021/cb3002478

569

29.

McCarthy, J. K., Smith, S. R., McCrow, J. P., Tan, M., Zheng, H., Beeri, K., Roth, R.,

570

Lichtle, C., Goodenough, U., Bowler, C. P., Dupont, C. L., and Allen, A. E. (2017)

571

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and

572

Drives Repartitioning of Carbon Flux in a Model Pennate Diatom, The Plant Cell 29,

573

2047-2070 DOI: 10.1105/tpc.16.00910

574

30.

Fernández, E., Schnell, R., Ranum, L. P., Hussey, S. C., Silflow, C. D., and Lefebvre, P.

575

A. (1989) Isolation and characterization of the nitrate reductase structural gene of

576

Chlamydomonas reinhardtii, Proceedings of the National Academy of Sciences of the

577

United States of America 86, 6449-6453 DOI:

578

31.

Iwanaga, S., Kato, T., Kaneko, I., and Yuda, M. (2012) Centromere plasmid: a new

579

genetic tool for the study of Plasmodium falciparum, PLoS ONE 7, e33326 DOI:

580

10.1371/journal.pone.0033326

581

32.

Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li,

582

Y., Fine, E. J., Wu, X., Shalem, O., Cradick, T. J., Marraffini, L. A., Bao, G., and Zhang,

583

F. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases, Nat Biotechnol 31,

584

827-832 DOI: 10.1038/nbt.2647 20


Page 21 of 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

585


33.

Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M., and Valen, E. (2014)

586

CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing, Nucleic Acids

587

Res 42, W401-407 DOI: 10.1093/nar/gku410

588 589

34.

Hong, H.-Y., Yoo, G.-S., and Choi, J.-K. (2002) Detection of Proteins on Blots Using Direct Blue 71, 0, 387-392 DOI: 10.1385/1-59259-169-8:387

590 591 592 593 594

21



TOC graphic 39x19mm (300 x 300 DPI)


Page 22 of 25

Page 23 of 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


Figure 1. A one-vector CRISPR system for gene disruption in N. oceanica. (A) The pNOC-CRISPR vector series includes a Hygromycin B resistance cassette (HygC, green). A bidirectional promoter (Ribi) drives the transcription of the Cas9-reporter fusion and gRNA scaffold (scaffold, blue), along with the LDSP and CS terminators (LDSP-T, CS-T) respectively. Cas9 is fused to either the GFP or Nlux (with HA tag) reporters by a 3x glycine-serine linker (GSGSGS) and contains SV40 nuclear localization signals on the N’ and C’ termini (NLS). The gRNA scaffold and the 3’ self-cleaving HDV ribozyme is integrated into the vector. The 5’ hammerhead ribozyme (HH) specific for each guide sequence (GS, orange) is fused to the gRNA scaffold (scaffold) to form a sgRNA. Ribozymes are highlighted in yellow. Unique restriction sites are shown with an upwards line and the name in italics. (B) Confocal analysis of DAPI nuclear staining, Cas9-GFP signal, and merged brightfield, DAPI and GFP signal in N. oceanica cells. Scale bar of 2 µM. (C) Immunoblotting with an α-GFP antibody detected the Cas9-GFP produced in N. oceanica transformed with pNOC-CRISPR-GFP. A N. oceanica line producing a delta-5 fatty acid desaturase (~75 kDa) fused with CFP was used as a GFP positive control (+). (D) Immunoblotting with an α-HA antibody detected the appropriately sized Cas9-Nlux-HA in N. oceanica transformed with pNOC-CRISPR. Wild-type N. oceanica was included as a negative control (WT). For (C) and (D) numbers on the left of immunoblots indicate size markers (KDa).

60x45mm (300 x 300 DPI)



Figure 2 99x124mm (300 x 300 DPI)


Page 24 of 25

Page 25 of 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


Figure 3 73x67mm (300 x 300 DPI)


Nontransgenic Marker-Free Gene Disruption by an ... - ACS Publications

Recommend Documents