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Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda
Journal Pre-proof CRISPR/Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda Yuying Sun, Congcong Yan, Mengfei Liu, Yujie Liu, Wenzheng Wang, Wei Cheng, Fusheng Yang, Jiquan Zhang PII:
S1050-4648(19)31166-0
DOI:
https://doi.org/10.1016/j.fsi.2019.12.037
Reference:
YFSIM 6679
To appear in:
Fish and Shellfish Immunology
Received Date: 23 October 2019 Revised Date:
9 December 2019
Accepted Date: 13 December 2019
Please cite this article as: Sun Y, Yan C, Liu M, Liu Y, Wang W, Cheng W, Yang F, Zhang J, CRISPR/ Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.037. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphical Abstract
1
CRISPR/Cas9-mediated deletion of one carotenoid isomerooxygenase
2
gene (EcNinaB-X1) from Exopalaemon carinicauda
3 Yuying Sun a, Congcong Yan a, Mengfei Liu a, Yujie Liu a, Wenzheng Wang a, Wei Cheng a,
4
Fusheng Yang b, Jiquan Zhang a,
5
b, *
6 a
7
Laboratory of Zoological Systematics and Application of Hebei Province, College of Life
8
Sciences, Hebei University, Baoding 071002, China
9
b
Xiaoshan Donghai Aquaculture Co., Ltd, Xiaoshan 310012, China
11
*
Corresponding author:
12
E-mail: [email protected]
10
13 14 15
Running title: CRISPR/Cas9-mediated deletion of EcNinaB-X1 gene
16
Abstract During the immune defense reaction of invertebrate, a plenty of reactive oxygen species
17
(ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of
18
these reactive molecules in animals will cause serious cell damage. Carotenoids could function as
19
scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid
20
isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda.
21
EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns
22
were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed.
23
Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first
24
exon of EcNinaB-X1 gene, 26 EcNinaB-X1-KO prawns were generated and the mutant rate
25
reached 6.5% after embryo injection. The EcNinaB-X1-KO prawns had significant lower mortality
26
than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or
27
Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid
28
isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable
29
gene mutagenesis.
30 31 32
Keywords: Exopalaemon carinicauda; carotenoid isomerooxygenase; CRISPR/Cas9
33
1. Introduction
34
In 2013, clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR
35
associated protein (Cas9) technology was reported to be used in the genome editing of organisms
36
[1, 2]. Since then, CRISPR/Cas9 technology has been widely used in the genome editing of many
37
organisms including mammals [2-4], fly fruit [5, 6], zebrafish [7, 8], prawn [9-11] and so on. It
38
can produce targeted double-strand breaks (DSBs) in the genome and the resulting DSBs are
39
repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathway,
40
thereby causing mutations [12].
41
A lot of crustaceans, such as crayfish, crabs, lobsters, prawns, and shrimp, are important
42
aquaculture species. At present, the genome draft of some crustaceans including Litopenaeus
43
vannamei [13], Exopalaemon carinicauda [14], Neocaridina denticulate [15], and Eriocheir
44
sinensis [16] has been reported. CRISPR/Cas9 technology was a useful tool to clarify the function
45
of genes by loss-of-function approaches in vivo. The ridgetail white prawn E. carinicauda, one of
46
the important commercial shrimp species, naturally distributed in the coasts of China [9]. In our
47
previous research, we used it as a model animal of crustacean in basic research and successfully
48
performed the genome editing using CRISPR/Cas9 technology [11]. We developed a highly
49
efficient microinjection method in E. carinicauda and deleted some interest genes using
50
CRISPR/Cas9 technology [9, 10, 17]. In addition, the low-coverage sequencing and de novo
51
assembly of E. carinicauda genome had also been finished, which made it possible to promote the
52
basic research of crustaceans [14].
53
Carotenoids such as beta-carotene, lycopene, lutein and β-cryptoxanthine are produced in
54
plants, certain bacteria, algae and fungi, where they function as accessory photosynthetic pigments
55
and as scavengers of oxygen radicals for photoprotection [18]. Recently, since carotenoid
56
accumulation played a key role in the formation of colorful animals, more researches have focused
57
on the change of body color in aquaculture animals [19]. All animals cannot produce these
58
naturally-occurring carotenoids, so they must obtain them from their diet. There are some
59
carotenoid oxygenases, including carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
60
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2), which can cleave a
61
variety of carotenoids into a range of biologically important products [20, 21]. Then, the cleaved
62
products function as hormones, pigments, flavors, floral scents and defense compounds in animals.
63
In addition, during the immune defense reaction of invertebrate, a plenty of reactive oxygen
64
species (ROS) could be induced to product. Though ROS can kill foreign invaders, the
65
accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids
66
could function as scavengers of oxygen radicals [22-24].
67
At present, there is no report about the function of carotenoid oxygenase in decapods. In this
68
research, the full-length cDNA sequence of one carotenoid isomerooxygenase gene (named
69
EcNinaB-X1) was cloned from E. carinicauda. Then, EcNinaB-X1 knock-out (EcNinaB-X1-KO)
70
prawns were produced via CRISPR/Cas9 technology and the change of phenotypes was analyzed.
71
Furtherly, the EcNinaB-X1-KO prawns were challenged with different pathogenic bacteria and the
72
function of EcNinaB-X1 in immune defense was also clarified.
73 74
2. Materials and methods
75
2.1. Ethical Statement
76
All efforts were made to minimize animal suffering. This article does not contain any studies
77
with human participants.
78 79
2.2 Cultivation of the experimental animals The ridgetail white prawns, E. carinicauda, were bred in plastic tanks filled with aerated fresh
80 81
seawater at 26 ºC, and fed twice per day in our laboratory.
82
Referring to our previous research [11], the one-cell stage embryos were collected from the
83
randomly selected spawning prawns and transferred to a clean petri dish loaded up with
84
appropriate filter-sterilized seawater before microinjection. After being injected, the experimental
85
embryos were put in the clean petri dishes containing sterilized seawater and incubated on a
86
shaking bath at 26 ºC with the speed of 100 rpm. Fresh clean seawater was changed per day. The
87
mysis larval prawns were fed with Artemia salina larvae and the juvenile prawns were fed with
88
bait.
89 90
2.3. RNA isolation, cDNA synthesis and bioinformatic analysis
91
Fifteen healthy prawns with a body length of 4.0 ± 0.5 cm were collected for tissue
92
distribution analysis. Hepatopancreas, muscle, eyestalk, gills, nerve, intestines, epidermis and
93
heart were dissected out and immediately preserved in liquid nitrogen for RNA extraction.
94
Total RNA was extracted from the collected samples with Trizol® reagent (Thermo, USA).
95
Then, the extracted RNA was treated with RQI RNase-Free DNase (Promega, USA). Two
96
micrograms of total RNA and 0.2 µM random hexamer primers were used to synthesize cDNA by
97
M-MLV reverse transcriptase (Promega, USA).
98
Based on the transcriptomic and genomic data of E. carinicauda [14], the full-length
99
NinaB-X1
sequence
of
E.
carinicauda
(EcNinaB-X1)
was
confirmed
by
reverse
100
transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence and deduced amino
101
acid
102
(http://www.ncbi.nlm.nih.gov/BLAST/). The characteristic structure of deduced EcNinaB-X1 was
103
predicted by SMART program (http://smart.embl-heidelberg.de/). The multiple sequence
104
alignments and phylogenetic analysis were performed using CLUSTAL W and MEGA 7.0 [25].
sequence
of
EcNinaB-X1
were
analyzed
by
BLAST
on-line
105 106
2.4. EcNinaB-X1 expression in different tissues analyzed by quantitative real-time PCR
107
Epidermis, heart, gill, eyestalk, hepatopancreas, intestine, stomach, nerve and muscle were
108
separated from 15 healthy prawns with a body length of 4.0 ± 0.5 cm for tissue distribution.
109
Quantitative real-time PCR (qRT-PCR) [26] was used to analyze EcNinaB-X1 distribution in
110
different tissues of E. carinicauda using Mastercycler ep realplex (Eppendorf). 18S rRNA was
111
used as the internal control. Primers were shown in Table 1. The expected size of EcNinaB-X1 and
112
18S rRNA was 131 bp and 147 bp, respectively. Samples were run in triplicate on the PCR System.
113
The data were analyzed using the comparative CT method and then subjected to one-way ANOVA
114
using SPSS 19.0. The p values less than 0.05 were considered statistically significant.
115 116 117 118
2.5. Designation and synthesis of gRNA specialized for EcNinaB-X1 According to our previous research [11], we designed one sgRNA target site for EcNinaB-X1 with the online tool ZiFiT (http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx).
119
The gRNA of EcNinaB-X1 was synthesized using the Thermo Scientific TranscriptAid T7
120
High Yield Transcription Kit (Thermo, USA). Then, it was purified by phenol chloroform
121
extraction. The gRNA concentration was assessed by Nanodrop 2000 (Thermo Fisher Scientific,
122
USA) and the quality was assessed by electrophoresis on 1% agarose gel. Then it was preserved at
123
-80 ºC in portions for microinjection.
124 125
2.6. Preparation of Cas9 mRNA
126
According to our previous research [11], the pCMV-Cas9 plasmid (Sigma-Aldrich, USA)
127
was linearized by Xba I (Takara, Dalian) and purified by ethanol precipitation. The linearized
128
product was used as the template to synthesize Cas9 mRNA that have both 5’cap and 3’poly (A)
129
tail in vitro with mMACHINE® T7 Ultra Kit (Ambion, USA). Then, it was purified by phenol
130
chloroform extraction and preserved at -80 ºC in portions for microinjection.
131 132
2.7. Microinjection and Indels detection by Sanger sequencing
133
The microinjection liquid contains 200 ng/µL Cas9 mRNA, 100 ng/µL gRNA and 0.05% of
134
the inert dye phenol red as the indicator in the buffer (100 mM HEPES, 1.5 M NaCl). Before
135
microinjection, the liquids were filtered through 0.22 µm filtering membranes. A Warner
136
PLI-100A Pico-Injector microinjector (Warner Instruments, USA) and a dissecting microscope
137
MN-152 micromanipulator (Narishige, Japan) were used for microinjection with standardized
138
Femtotip II sterile microcapillaries (Eppendorf, Germany). The injection volume was
139
approximately 0.5 nL. The prawns in experimental group were injected with Cas9 mRNA and
140
EcNinaB-X1 gRNA.
141
The genomic DNA of mysis larvae prawns was extracted and the genomic region flanking the
142
target site was amplified by MightyAmp® Genotyping Kit (Takara, Dalian) according to the
143
manufacturer’s instruction. For Sanger sequencing detection, the amplified PCR products were
144
purified using Gel Extraction Kit (Tiangen, China) and then cloned into pMD19-T Simple Vector
145
(Takara, Dalian). The detection primers used to amplify the target fragment are detEcNinaB-F and
146
detEcNinaB-R (Table 1).
147 148
2.8. Evaluation of CRISPR/Cas9 generated EcNinaB-X1-KO prawns
149
According to our previous research [17], the heterozygous EcNinaB-X1-KO progenies from
150
the same family with one wild-type allele and one allele harboring 4 bp deletions at exon 1 were
151
crossed to produce wild-type, heterozygous and homozygous EcNinaB-X1-KO prawns [11]. The
152
generated wild-type prawns were crossed to produce the filial generations (wild-type prawns) and
153
the generated homozygous EcNinaB-X1-KO prawns were also crossed to produce the filial
154
generations (EcNinaB-X1-KO prawns). After being hatched, the prawns were fed with Artemia
155
salina larvae. When the mysis larvae grew into juvenile prawns, they were fed with clam meat.
156
Difference of phenotype between the wild-type and EcNinaB-X1-KO prawns: the prawns and
157
dissected hepatopancreas were photographed and the color difference was calculated according to
158
the method described by reference [27].
159
Vibrio parahaemolyticus and Aeromonas hydrophila used in this study were isolated and
160
identified by Dr. Yuying Sun. The bacteria were routinely cultured in Tryptic Soy Broth (TSB,
161
Difco) or TSA medium supplemented with additional 1% NaCl at 28 ºC, 180 rpm.
162
The wild-type and EcNinaB-X1-KO prawns cultured with the same size were challenged with
163
V. parahaemolyticus and A. hydrophila according to previous research [17]. EcNinaB-X1-KO
164
group and wild-type group were set up for each sampling point and 200 prawns were sampled
165
from each group. For the bacterial challenge experiment, the experimental group was injected
166
individually with 10 µL phosphate buffer saline (PBS) containing V. parahaemolyticus or A.
167
hydrophila (107 CFU mL-1). At the same time, the prawns injected with 10 µL sterile PBS were
168
maintained as the control. The residual prawns were calculated at 0, 12, 24, 48, 72, 96, and 120 h.
169 170
3. Results
171
3.1. Amplification and characterization of EcNinaB-X1
172
Based on the transcriptomic and genomic data of E. carinicauda published by Yuan et al. [14],
173
the full-length cDNA sequence of EcNinaB-X1 was obtained with 3185 bp (GenBank accession no.
174
MN583041). As shown in Fig.1, the nucleotide sequence of EcNinaB-X1 contained a 2070 bp
175
open reading frame (ORF) encoding EcNinaB-X1 of 689 amino acids with a predicted molecular
176
weight (MW) about 78286.67 Da and theoretical isoelectric point (pI) of 7.37. No putative signal
177
peptide was found. The domain architecture of deduced EcNinaB-X1 predicted by SMART
178
software showed that there was a RPE65 domain (residues 152-670) (Fig. 2). In addition, the
179
genomic DNA fragment of EcNinaB-X1 with the corresponding cDNA sequence was obtained,
180
which showed that it was composed of 12 exons and 11 introns (Fig.3). All intron-exon boundaries
181
are consistent with the consensus splicing junctions at both the 5’ splice donor site (GT) and the 3’
182
splice acceptor site (AG) of each intron.
183
A multiple sequence alignment showed that EcNinaB-X1 displayed high identities with
184
carotenoid isomerooxygenase of Armadillidium vulgare (AvNinaB-X1, 60%), and Nilaparvata
185
lugens (NlNinaB-X1, 51%) (Fig. 4).
186
Amino acid sequences of carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
187
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2) from different species
188
were collected from the NCBI database and a phylogenetic tree was constructed using
189
Neighbor-joining method. The phylogenetic analysis showed that EcNinaB-X1 was divided into
190
the same branch of NinaB (Fig.5).
191 192
3.2 Tissue distribution of EcNinaB-X1 mRNA
193
Expression profiling of EcNinaB-X1 mRNA in different tissues of E. carinicauda was
194
detected by quantitative real-time PCR. The expression of EcNinaB-X1 was expressed
195
constitutively in all of the detected tissues and highly expressed in the hepatopancreas, gill, and
196
stomach (Fig. 6).
197 198
3.3. Knockout of EcNinaB-X1 using CRISPR/Cas9 technology
199
Ten days after injection to the one-cell embryo, ten embryos were selected randomly in each
200
group and extracted the genomic DNA. Then the target fragment was amplified with detection
201
primers. The sequencing results of the purified PCR products showed that multiple peaks occurred
202
initially after the PAM sites compared with blank and control groups (Fig. 7). It indicated that the
203
genome of prawns in the experimental group had been successfully edited. In order to identify the
204
result, cloning and Sanger sequencing were carried out. Results showed that seven types of
205
deletion mutation were generated in total, but no insertions were found (Fig. 8).
206
The mutation rate of the embryos injected with Cas9 mRNA and one sgRNA was analyzed
207
and compared with the results of our previous research (Table 2). Of the 400 injected one-cell
208
stage embryos with Cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 58
209
embryos could develop to postlarvae and the reproductive survival rate was 14.5%. Fifty days
210
later, when the 58 lived embryos developed to adult E. carinicauda, one leg of them was collected
211
respectively to detect indels and the results indicated that the number of mutant prawns was 26
212
and the mutant rate reached 6.5%. Furtherly, of the 26 mutant prawns, 5 kinds of mutant types
213
were identified in the targeted locus (Fig. 9).
214 215
3.4. Evaluation of CRISPR/Cas9 generated EcNinaB-X1-KO prawns
216
To determine the effect of the EcNinaB-X1-KO on the prawns, the phenotypes and dissected
217
hepatopancreas of wild-type and EcNinaB-X1-KO prawns were compared. The hepatopancreas
218
from EcNinaB-X1-KO prawns exhibited a red color compared with wild-type individuals (Fig.
219
10A, B). According to the reference [27], the industrial color criteria was used to evaluated the
220
color change in the hepatopancreas between wild-type and EcNinaB-X1-KO prawns, and the
221
EcNinaB-X1-KO prawns displayed a significant shift to red color compared to the wild-type
222
groups (Fig. 10C).
223
The effects of EcNinaB-X1-KO on the mortality of bacteria-challenged prawns by
224
challenging them with were also evaluated. The prawns in EcNinaB-X1-KO group had
225
significantly low mortality than those in wild-type group when the prawns were challenged with V.
226
parahaemolyticus or A. hydrophila (Fig. 11A, B). For the Vibrio-challenged prawns, the mortality
227
reached 50% in wild-type group and only 20% in EcNinaB-X1-KO group at 48 h post-challenge
228
and there was significant difference between wild-type and EcNinaB-X1-KO prawns (p < 0.01).
229
For the Aeromonas-challenged prawns, the mortality reached 65% in wild-type group and only 25%
230
in EcNinaB-X1-KO group at 48 h post-challenge and there was significant difference between
231
wild-type and EcNinaB-X1-KO prawns (p < 0.05). The negative control showed a cumulative
232
mortality of ~10% indicating that PBS-injection itself was non-toxic in prawns. Overall, these
233
results indicated that the deletion of EcNinaB-X1 in E. carinicauda decreased prawn’s mortality
234
following V. parahaemolyticus or A. hydrophila challenge.
235 236
4. Discussion
237
Carotenoids are naturally occurring red, orange and yellow pigments that are synthesized by
238
plants, alga and some microorganisms and fulfill many important physiological functions [28].
239
Carotenoids are fat-soluble compounds, and it is important to enhance the bioavailability of
240
β-carotene from food or supplements in animals [29]. β-carotene is involved in a number of
241
beneficial functions in animals such as taking part in crucial signaling functions of its metabolites
242
and working as an antioxidant [27]. In addition, the accumulation of carotenoids in animals could
243
change the fat color in some animals, which make it important in animal breeding [27, 30]. Li et al
244
[31] cultured a new variety of Yesso scallop (Patinopecten yessoensis) with orange adductor
245
muscle, the ‘Haida golden scallop’, which was caused by carotenoid accumulation.
246
In animals, carotenoids could be cleaved into a range of biologically important products and
247
carotenoid oxygenases play an important role in the reaction [32]. It is reported that there are some
248
carotenoid oxygenases, including carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
249
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2) [20, 21]. In this
250
research, the full-length cDNA and genomic DNA of one carotenoid isomerooxygenase gene
251
(EcNinaB-X1) were obtained from E. carinicauda. There is one typical RPE65 domain at the
252
position of 152-670 in the deduced EcNinaB-X1 amino acid sequence. From the bioinformatic
253
analysis, we could find that the RPE65 domain played the oxidoreductase activity acting on single
254
donors with incorporation of molecular oxygen, or two atoms of oxygen [29]. Among three typical
255
carotenoid oxygenases, the phylogenetic analysis showed that EcNinaB-X1 was divided into the
256
same branch of NinaB, not belonging to β, β-carotene 15, 15'-monooxygenase (BCMO) and β,
257
β-carotene 9', 10'-oxygenase (BCO2).
258
Niu et al [27] reported that the biallelic modification of BCO2 from sheep resulted in yellow
259
fat, compared with the fat color in wild types (snow-flower white). Vage & Boman [33] reported
260
that a nonsense mutation in the BCO2 gene was tightly associated with accumulation of
261
carotenoids in adipose tissue of sheep (Ovis aries). Strychalski et al [34] reported that an
262
AAT-deletion mutation in the coding sequence of the BCO2 gene was related to the formation of
263
yellow-fat rabbit. Kyle-Little et al [35] reported that the mutant of BCMO1 gene affected macular
264
pigment optical density in young healthy caucasians. Amengual et al [36] reported that the
265
BCMO1 acted as a critical molecular player in deducing body adiposity of mice. However, there
266
was no report about the biological function of NinaB in regulating the physiology and
267
biochemistry of animals. In this research, we used CRISPR-Cas9 system to disrupt EcNinaB-X1
268
gene and obtained wild-type and homozygous mutants at EcNinaB-X1 loci in the offspring. In this
269
study, wild-type and homozygous EcNinaB-X1-KO prawns were selected as the experimental
270
animals to study the function of EcNinaB-X1 in immune defense. EcNinaB-X1-KO prawns
271
showed much lower mortality than those in wild-type group after V. parahaemolyticus or A.
272
hydrophila challenge, which indicated that EcNinaB-X1 might play a key role in immune defense
273
of prawns. In addition, the deletion of EcNinaB-X1 gene resulted in the color shift compared with
274
wild-type prawns. From above research, we concluded that NinaB-X1 gene could be used as a
275
candidate gene in marker-assistant selection in animal breeding.
276 277 278
Conflict of interest There is no conflict of interest.
279 280
Acknowledgments
281
This work was supported by the National Key R&D Program of China (No.
282
2018YFD0900205), National Natural Science Foundation of China (Grant Nos. 31872613,
283
41876196), The Natural Science Foundation of Hebei Province of China (Grant Nos.
284
C2019201236, D2019201239), Innovation & entrepreneurship training program for college
285
students of Hebei Province (Grant No. S201910075061), Innovation & entrepreneurship training
286
program for college students of Hebei University (Grant No. 2019177)， and Hangzhou Qianjiang
287
Special Expert for Jiquan Zhang. We are grateful to Dr. Huan Gao for providing the prawns.
288 289
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activity, Computational and Theoretical Chemistry 1077 (2016) 92-8. [23] T. Papa, V. Pinho, E. do Nascimento, W. Santos, A. Burtoloso, L. Skibsted, D. Cardoso, Astaxanthin diferulate as a bifunctional antioxidant, Free Radical Res. 49(1) (2015) 102-11. [24] E. Rodrigues, L. Mariutti, A. Mercadante, Scavenging capacity of marine carotenoids against reactive oxygen and nitrogen species in a membrane-mimicking system, Mar. Drugs 10(8) (2012) 1784-98. [25] S. Kumar, G. Stecher, K. Tamura, MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets, Mol Biol Evol 33(7) (2016) 1870-74. [26] C. Yang, J. Zhang, F. Li, H. Ma, Q. Zhang, T. Jose Priya, X. Zhang, J. Xiang, A Toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection, Fish Shellfish Immunol 24(5) (2008) 564-74. [27] Y. Niu, M. Jin, Y. Li, P. Li, J. Zhou, X. Wang, B. Petersen, X. Huang, Q. Kou, Y. Chen, Biallelic beta-carotene oxygenase 2 knockout results in yellow fat in sheep via CRISPR/Cas9, Anim Genet 48(2) (2016) 242-44. [28] J. Alcaino, M. Baeza, V. Cifuentes, Carotenoid Distribution in Nature, Subcell. Biochem. 79 (2016) 3-33. [29] J.D. Ribaya-Mercado, Influence of dietary fat on beta-carotene absorption and bioconversion into vitamin A, Nutr. Rev. 60(4) (2002) 104-10. [30] K.M. Sefc, A.C. Brown, E.D. Clotfelter, Carotenoid-based coloration in cichlid fishes, Comp. Biochem. Physiol. A Mol. Integr. Physiol. 173C (2014) 42-51. [31] X. Li, X. Ning, J. Dou, Q. Yu, S. Wang, L. Zhang, S. Wang, X. Hu, Z. Bao, An SCD gene from the Mollusca and its upregulation in carotenoid-enriched scallops, Gene 564(1) (2015) 101-8. [32] G. Giuliano, S. Al-Babili, J. von Lintig, Carotenoid oxygenases: cleave it or leave it, Trends Plant Sci. 8(4) (2003) 145-9. [33] D.I. Vage, I.A. Boman, A nonsense mutation in the beta-carotene oxygenase 2 (BCO2) gene is tightly associated with accumulation of carotenoids in adipose tissue in sheep (Ovis aries), BMC Genet. 11 (2010) 10. [34] J. Strychalski, P. Brym, U. Czarnik, A. Gugolek, A novel AAT-deletion mutation in the coding sequence of the BCO2 gene in yellow-fat rabbits, J Appl Genet 56(4) (2015) 535-37. [35] Z. Kyle-Little, A.J. Zele, C.P. Morris, B. Feigl, The effect of BCMO1 gene variants on macular pigment optical density in young healthy caucasians, Front Nutr 1 (2014) 22. [36] J. Amengual, E. Gouranton, Y.G. van Helden, S. Hessel, J. Ribot, E. Kramer, B. Kiec-Wilk, U. Razny, G. Lietz, A. Wyss, A. Dembinska-Kiec, A. Palou, J. Keijer, J.F. Landrier, M.L. Bonet, J. von Lintig, Beta-carotene reduces body adiposity of mice via BCMO1, PLoS ONE 6(6) (2011) e20644.
Table 1 Primers mentioned in the paper
383
Primers
384 385
Sequences (5’- 3’)
Sequence information
EcNinaF
ACAGCCCCTTGTCGTGTCCGT
Real-time PCR
EcNinaR
TCGTCGCTCCCGTGTCCCT
Real-time PCR
18S-F
TATACGCTAGTGGAGCTGGAA
Real-time PCR
18S-R
GGGGAGGTAGTGACGAAAAAT
Real-time PCR
DetEcNinaF
TTTCAGTAACTCTTACGATTC
Detection of mutation
DetEcNinaR
CCCGCAGTGGTAGGAATAGG
Detection of mutation
Note: F and R stand for forward primers and reverse ones, respectively.
Table 2 Mutation frequencies induced by microinjection of Cas9 mRNA and EcNinaB-X1 gRNA
386
Injected Group
Survival
Mutant
Survival
Mutant
RNA concentration
100 ng/µL
Embryos
Postlarvae
Postlarvae
Rate
Rate
247
35
18
14.17%
7.29%
[11]
250
23
12
9.20%
4.80%
[9]
400
58
26
14.50%
6.50%
In this paper
200 ng/µL
EcChi4 (gRNA-EcChi4)
(pCMV-Cas9)
100 ng/µL
200 ng/µL
EcMIH (gRNA-EcMIH)
(pCMV-Cas9)
100 ng/µL
200 ng/µL
EcNinaB-X1 (gRNA-EcNinaB-X1)
References
(pCMV-Cas9)
387
Legends of Figures
388
Fig.1 Nucleotide and deduced amino acid sequences of EcNinaB-X1 gene.
389
Nucleotides are numbered on the both sides of the sequence. The letters marked with double
390
underline represented the RPE65 domains.
391
Fig.2 The domain architecture of deduced EcNinaB-X1.
392
Fig. 3 Schematic representation of the structure of EcNinaB-X1 gene depicting exons, gRNA
393
target sequence (sequence in blue at the bottom) and PAM site (in red). The numbers indicate the
394
exact lengths of the exons.
395
Fig. 4 Alignment of the amino acid sequence of EcNinaB-X1 with other known NinaBs.
396
The identical residues are shown in solid boxes. Sequences start at the first methionine residue.
397
Armadillidium vulgare (AvNinaB-X1, GenBank accession no. RXG54508.1); Nilaparvata lugens
398
(NlNinaB-X1, XP_022185479.1); E. carinicauda (EcNinaB-X1, MN583041, in this research).
399
Fig.5 Phylogenetic tree of carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
400
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2) from different species
401
based on the amino acid sequence comparisons.
402
Aedes albopictus (AaNinaB-X1, GenBank accession no. XP_019545564.1); Aethina tumida
403
(AtNinaB-X1, XP_019870879.1); Anoplophora glabripennis (AgNinaB-X1, XP_023310515.1);
404
Apis cerana (AcNinaB-X1, XP_016913671.1); A. dorsata (AdNinaB-X1, XP_006611100.1); A.
405
florea
406
Armadillidium vulgare (AvNinaB-X1, GenBank accession no. RXG54508.1); Bombus terrestris
407
(BtNinaB-X1, XP_003400564.1); Branchiostoma belcheri (BbBCO2-X1, XP_019636508.1);
408
Callorhinchus milii (CmBCMO-X1, XP_007887463.1); Ceratina calcarata (CcNinaB-X1,
(AfNinaB-X1,
XP_003695005.1);
A.
mellifera
(AmNinaB-X1,
XP_394000.4);
409
XP_017875448.1); Clupea harengus (ChBCO2-X1, XP_012685829.1); Dendroctonus ponderosae
410
(DpNinaB-X1, XP_019773593.1); Diachasma alloeum (DaNinaB-X1, XP_015113077.1);
411
Dinoponera quadriceps (DqNinaB-X1, XP_014471901.1); Dufourea novaeangliae (DnNinaB-X1,
412
XP_015439200.1); Eufriesea mexicana (EmNinaB-X1, XP_017759905.1); Fopius arisanus
413
(FaNinaB-X1, XP_011310428.1); Fundulus heteroclitus (FhBCMO-X1, XP_012709399.1);
414
Habropoda laboriosa (HlNinaB-X1, XP_017798623.1); Harpegnathos saltator (HsNinaB-X1,
415
XP_011149992.1); Ictalurus punctatus (IpBCO2-X1, XP_017316112.1); Latimeria chalumnae
416
(LcBCMO-X1,
417
XP_014345540.1;
418
XP_013396242.1); Megachile rotundata (MrNinaB-X1, XP_003702796.1); Nilaparvata lugens
419
(NlNinaB-X1, XP_022185479.1); Pseudomyrmex gracilis (PgNinaB-X1, XP_020278007.1);
420
Salmo salar (SsBCMO-X1, XP_014030705.1; SsBCMO-X2, XP_014030708.1; SsBCMO-X3,
421
XP_014030708.1); Sarcophilus harrisii (ShBCMO-X1, XP_003757903.1); Trichogramma
422
pretiosum (TpNinaB-X1, XP_014224940.1); E. carinicauda (EcNinaB-X1, MN583041, in this
423
research). Values on the line are bootstrap values showing percentage confidence of relatedness.
424
Fig.6 Detection of EcNinaB-X1 transcripts in different tissues of E. carinicauda. Tissues were
425
shown in the abscissa. The amount of EcNinaB-X1 mRNA was normalized to the 18S rRNA
426
transcript level. Data are shown as means ± SD (standard deviation) of three separate individuals
427
in the tissues.
428
Fig.7 Sanger sequencing of the PCR products from 10 prawns indicated the indel mutations
429
caused by CRISPR/Cas9 genome editing system. WT means the wild-type prawns (blank group).
430
M1 and M2 mean injected embryos. The PAM site was represented in pink rectangles.
XP_006004911.1; LcBCO2-X3,
LcBCO2-X1, XP_014345541.1);
XP_014345536.1; Lingula
anatina
LcBCO2-X2, (LaBCO2-X1,
431
Fig.8 Sequencing of CRISPR/Cas9 treated prawns. The wild-type sequence (ref) is shown at the
432
top. The gRNA site is underlined and the sequence in pink frame (TGG) represents the PAM
433
sequence. The numbers on the right represents the number of mutations recovered by sequencing.
434
Within the sequences, deletions are indicated by dashed lines.
435
Fig. 9 Sanger sequencing of the PCR products from prawns individually indicated the indel
436
mutations caused by CRISPR/Cas9 genome editing system. WT means the wild-type prawn (blank
437
group). MT-1, 2, 3, 4, and 5 mean five mutant-type prawns. The PAM site was represented in
438
black rectangles.
439
Figure 10 Phenotypes and dissection of EcNinaB-X1 knockouts in the hepatopancreas of E.
440
carinicauda. (a) Phenotypes of wild-type and EcNinaB-X1-KO prawns. (b) Hepatopancreas
441
dissected from prawns showed in figure (a). (c) Color differences in the hepatopancreas of
442
wild-type and EcNinaB-X1-KO prawns; ‘R’ indicates red color, ‘G’ indicates green color, ‘B’
443
indicates blue color. According to the reference [27], we defined the (R+G)/B value to indicate the
444
red color.
445
Fig. 11 The mortality of the prawns in wild-type group and EcNinaB-X1-KO group after the
446
prawns were challenged with Vibrio parahaemolyticus and equal volume of PBS (A), or
447
Aeromonas hydrophila and equal volume of PBS (B) at 0, 12, 24, 48, 72, 96, and 120 h.
448
449
450 Fig.1
451 452 453
.
454 455 456
Fig.2
457 458 459 460
Fig. 3
461 462 463
Fig. 4
464 465 466
Fig.5
467 468 469
Fig.6
470
471 472 473
Fig.7
474 475 476
Fig. 8
477
478 479 480
Fig. 9
481
482
(a)
483
(b)
(c)
484 485
Figure 10
486
.
487
488 489
Fig. 11
Highlights
EcNinaB-X1 gene was cloned from E. carinicauda. The expression profiles of EcNinaB-X1 were demonstrated. CRISPR-Cas9 system efficiently generated indels in EcNinaB-X1 loci. EcNinaB-X1-KO prawns had lower mortality than wild-type after bacterial challenge.
S1050-4648(19)31166-0
DOI:
https://doi.org/10.1016/j.fsi.2019.12.037
Reference:
YFSIM 6679
To appear in:
Fish and Shellfish Immunology
Received Date: 23 October 2019 Revised Date:
9 December 2019
Accepted Date: 13 December 2019
Please cite this article as: Sun Y, Yan C, Liu M, Liu Y, Wang W, Cheng W, Yang F, Zhang J, CRISPR/ Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.12.037. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphical Abstract
1
CRISPR/Cas9-mediated deletion of one carotenoid isomerooxygenase
2
gene (EcNinaB-X1) from Exopalaemon carinicauda
3 Yuying Sun a, Congcong Yan a, Mengfei Liu a, Yujie Liu a, Wenzheng Wang a, Wei Cheng a,
4
Fusheng Yang b, Jiquan Zhang a,
5
b, *
6 a
7
Laboratory of Zoological Systematics and Application of Hebei Province, College of Life
8
Sciences, Hebei University, Baoding 071002, China
9
b
Xiaoshan Donghai Aquaculture Co., Ltd, Xiaoshan 310012, China
11
*
Corresponding author:
12
E-mail: [email protected]
10
13 14 15
Running title: CRISPR/Cas9-mediated deletion of EcNinaB-X1 gene
16
Abstract During the immune defense reaction of invertebrate, a plenty of reactive oxygen species
17
(ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of
18
these reactive molecules in animals will cause serious cell damage. Carotenoids could function as
19
scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid
20
isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda.
21
EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns
22
were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed.
23
Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first
24
exon of EcNinaB-X1 gene, 26 EcNinaB-X1-KO prawns were generated and the mutant rate
25
reached 6.5% after embryo injection. The EcNinaB-X1-KO prawns had significant lower mortality
26
than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or
27
Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid
28
isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable
29
gene mutagenesis.
30 31 32
Keywords: Exopalaemon carinicauda; carotenoid isomerooxygenase; CRISPR/Cas9
33
1. Introduction
34
In 2013, clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR
35
associated protein (Cas9) technology was reported to be used in the genome editing of organisms
36
[1, 2]. Since then, CRISPR/Cas9 technology has been widely used in the genome editing of many
37
organisms including mammals [2-4], fly fruit [5, 6], zebrafish [7, 8], prawn [9-11] and so on. It
38
can produce targeted double-strand breaks (DSBs) in the genome and the resulting DSBs are
39
repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathway,
40
thereby causing mutations [12].
41
A lot of crustaceans, such as crayfish, crabs, lobsters, prawns, and shrimp, are important
42
aquaculture species. At present, the genome draft of some crustaceans including Litopenaeus
43
vannamei [13], Exopalaemon carinicauda [14], Neocaridina denticulate [15], and Eriocheir
44
sinensis [16] has been reported. CRISPR/Cas9 technology was a useful tool to clarify the function
45
of genes by loss-of-function approaches in vivo. The ridgetail white prawn E. carinicauda, one of
46
the important commercial shrimp species, naturally distributed in the coasts of China [9]. In our
47
previous research, we used it as a model animal of crustacean in basic research and successfully
48
performed the genome editing using CRISPR/Cas9 technology [11]. We developed a highly
49
efficient microinjection method in E. carinicauda and deleted some interest genes using
50
CRISPR/Cas9 technology [9, 10, 17]. In addition, the low-coverage sequencing and de novo
51
assembly of E. carinicauda genome had also been finished, which made it possible to promote the
52
basic research of crustaceans [14].
53
Carotenoids such as beta-carotene, lycopene, lutein and β-cryptoxanthine are produced in
54
plants, certain bacteria, algae and fungi, where they function as accessory photosynthetic pigments
55
and as scavengers of oxygen radicals for photoprotection [18]. Recently, since carotenoid
56
accumulation played a key role in the formation of colorful animals, more researches have focused
57
on the change of body color in aquaculture animals [19]. All animals cannot produce these
58
naturally-occurring carotenoids, so they must obtain them from their diet. There are some
59
carotenoid oxygenases, including carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
60
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2), which can cleave a
61
variety of carotenoids into a range of biologically important products [20, 21]. Then, the cleaved
62
products function as hormones, pigments, flavors, floral scents and defense compounds in animals.
63
In addition, during the immune defense reaction of invertebrate, a plenty of reactive oxygen
64
species (ROS) could be induced to product. Though ROS can kill foreign invaders, the
65
accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids
66
could function as scavengers of oxygen radicals [22-24].
67
At present, there is no report about the function of carotenoid oxygenase in decapods. In this
68
research, the full-length cDNA sequence of one carotenoid isomerooxygenase gene (named
69
EcNinaB-X1) was cloned from E. carinicauda. Then, EcNinaB-X1 knock-out (EcNinaB-X1-KO)
70
prawns were produced via CRISPR/Cas9 technology and the change of phenotypes was analyzed.
71
Furtherly, the EcNinaB-X1-KO prawns were challenged with different pathogenic bacteria and the
72
function of EcNinaB-X1 in immune defense was also clarified.
73 74
2. Materials and methods
75
2.1. Ethical Statement
76
All efforts were made to minimize animal suffering. This article does not contain any studies
77
with human participants.
78 79
2.2 Cultivation of the experimental animals The ridgetail white prawns, E. carinicauda, were bred in plastic tanks filled with aerated fresh
80 81
seawater at 26 ºC, and fed twice per day in our laboratory.
82
Referring to our previous research [11], the one-cell stage embryos were collected from the
83
randomly selected spawning prawns and transferred to a clean petri dish loaded up with
84
appropriate filter-sterilized seawater before microinjection. After being injected, the experimental
85
embryos were put in the clean petri dishes containing sterilized seawater and incubated on a
86
shaking bath at 26 ºC with the speed of 100 rpm. Fresh clean seawater was changed per day. The
87
mysis larval prawns were fed with Artemia salina larvae and the juvenile prawns were fed with
88
bait.
89 90
2.3. RNA isolation, cDNA synthesis and bioinformatic analysis
91
Fifteen healthy prawns with a body length of 4.0 ± 0.5 cm were collected for tissue
92
distribution analysis. Hepatopancreas, muscle, eyestalk, gills, nerve, intestines, epidermis and
93
heart were dissected out and immediately preserved in liquid nitrogen for RNA extraction.
94
Total RNA was extracted from the collected samples with Trizol® reagent (Thermo, USA).
95
Then, the extracted RNA was treated with RQI RNase-Free DNase (Promega, USA). Two
96
micrograms of total RNA and 0.2 µM random hexamer primers were used to synthesize cDNA by
97
M-MLV reverse transcriptase (Promega, USA).
98
Based on the transcriptomic and genomic data of E. carinicauda [14], the full-length
99
NinaB-X1
sequence
of
E.
carinicauda
(EcNinaB-X1)
was
confirmed
by
reverse
100
transcription-polymerase chain reaction (RT-PCR). The nucleotide sequence and deduced amino
101
acid
102
(http://www.ncbi.nlm.nih.gov/BLAST/). The characteristic structure of deduced EcNinaB-X1 was
103
predicted by SMART program (http://smart.embl-heidelberg.de/). The multiple sequence
104
alignments and phylogenetic analysis were performed using CLUSTAL W and MEGA 7.0 [25].
sequence
of
EcNinaB-X1
were
analyzed
by
BLAST
on-line
105 106
2.4. EcNinaB-X1 expression in different tissues analyzed by quantitative real-time PCR
107
Epidermis, heart, gill, eyestalk, hepatopancreas, intestine, stomach, nerve and muscle were
108
separated from 15 healthy prawns with a body length of 4.0 ± 0.5 cm for tissue distribution.
109
Quantitative real-time PCR (qRT-PCR) [26] was used to analyze EcNinaB-X1 distribution in
110
different tissues of E. carinicauda using Mastercycler ep realplex (Eppendorf). 18S rRNA was
111
used as the internal control. Primers were shown in Table 1. The expected size of EcNinaB-X1 and
112
18S rRNA was 131 bp and 147 bp, respectively. Samples were run in triplicate on the PCR System.
113
The data were analyzed using the comparative CT method and then subjected to one-way ANOVA
114
using SPSS 19.0. The p values less than 0.05 were considered statistically significant.
115 116 117 118
2.5. Designation and synthesis of gRNA specialized for EcNinaB-X1 According to our previous research [11], we designed one sgRNA target site for EcNinaB-X1 with the online tool ZiFiT (http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx).
119
The gRNA of EcNinaB-X1 was synthesized using the Thermo Scientific TranscriptAid T7
120
High Yield Transcription Kit (Thermo, USA). Then, it was purified by phenol chloroform
121
extraction. The gRNA concentration was assessed by Nanodrop 2000 (Thermo Fisher Scientific,
122
USA) and the quality was assessed by electrophoresis on 1% agarose gel. Then it was preserved at
123
-80 ºC in portions for microinjection.
124 125
2.6. Preparation of Cas9 mRNA
126
According to our previous research [11], the pCMV-Cas9 plasmid (Sigma-Aldrich, USA)
127
was linearized by Xba I (Takara, Dalian) and purified by ethanol precipitation. The linearized
128
product was used as the template to synthesize Cas9 mRNA that have both 5’cap and 3’poly (A)
129
tail in vitro with mMACHINE® T7 Ultra Kit (Ambion, USA). Then, it was purified by phenol
130
chloroform extraction and preserved at -80 ºC in portions for microinjection.
131 132
2.7. Microinjection and Indels detection by Sanger sequencing
133
The microinjection liquid contains 200 ng/µL Cas9 mRNA, 100 ng/µL gRNA and 0.05% of
134
the inert dye phenol red as the indicator in the buffer (100 mM HEPES, 1.5 M NaCl). Before
135
microinjection, the liquids were filtered through 0.22 µm filtering membranes. A Warner
136
PLI-100A Pico-Injector microinjector (Warner Instruments, USA) and a dissecting microscope
137
MN-152 micromanipulator (Narishige, Japan) were used for microinjection with standardized
138
Femtotip II sterile microcapillaries (Eppendorf, Germany). The injection volume was
139
approximately 0.5 nL. The prawns in experimental group were injected with Cas9 mRNA and
140
EcNinaB-X1 gRNA.
141
The genomic DNA of mysis larvae prawns was extracted and the genomic region flanking the
142
target site was amplified by MightyAmp® Genotyping Kit (Takara, Dalian) according to the
143
manufacturer’s instruction. For Sanger sequencing detection, the amplified PCR products were
144
purified using Gel Extraction Kit (Tiangen, China) and then cloned into pMD19-T Simple Vector
145
(Takara, Dalian). The detection primers used to amplify the target fragment are detEcNinaB-F and
146
detEcNinaB-R (Table 1).
147 148
2.8. Evaluation of CRISPR/Cas9 generated EcNinaB-X1-KO prawns
149
According to our previous research [17], the heterozygous EcNinaB-X1-KO progenies from
150
the same family with one wild-type allele and one allele harboring 4 bp deletions at exon 1 were
151
crossed to produce wild-type, heterozygous and homozygous EcNinaB-X1-KO prawns [11]. The
152
generated wild-type prawns were crossed to produce the filial generations (wild-type prawns) and
153
the generated homozygous EcNinaB-X1-KO prawns were also crossed to produce the filial
154
generations (EcNinaB-X1-KO prawns). After being hatched, the prawns were fed with Artemia
155
salina larvae. When the mysis larvae grew into juvenile prawns, they were fed with clam meat.
156
Difference of phenotype between the wild-type and EcNinaB-X1-KO prawns: the prawns and
157
dissected hepatopancreas were photographed and the color difference was calculated according to
158
the method described by reference [27].
159
Vibrio parahaemolyticus and Aeromonas hydrophila used in this study were isolated and
160
identified by Dr. Yuying Sun. The bacteria were routinely cultured in Tryptic Soy Broth (TSB,
161
Difco) or TSA medium supplemented with additional 1% NaCl at 28 ºC, 180 rpm.
162
The wild-type and EcNinaB-X1-KO prawns cultured with the same size were challenged with
163
V. parahaemolyticus and A. hydrophila according to previous research [17]. EcNinaB-X1-KO
164
group and wild-type group were set up for each sampling point and 200 prawns were sampled
165
from each group. For the bacterial challenge experiment, the experimental group was injected
166
individually with 10 µL phosphate buffer saline (PBS) containing V. parahaemolyticus or A.
167
hydrophila (107 CFU mL-1). At the same time, the prawns injected with 10 µL sterile PBS were
168
maintained as the control. The residual prawns were calculated at 0, 12, 24, 48, 72, 96, and 120 h.
169 170
3. Results
171
3.1. Amplification and characterization of EcNinaB-X1
172
Based on the transcriptomic and genomic data of E. carinicauda published by Yuan et al. [14],
173
the full-length cDNA sequence of EcNinaB-X1 was obtained with 3185 bp (GenBank accession no.
174
MN583041). As shown in Fig.1, the nucleotide sequence of EcNinaB-X1 contained a 2070 bp
175
open reading frame (ORF) encoding EcNinaB-X1 of 689 amino acids with a predicted molecular
176
weight (MW) about 78286.67 Da and theoretical isoelectric point (pI) of 7.37. No putative signal
177
peptide was found. The domain architecture of deduced EcNinaB-X1 predicted by SMART
178
software showed that there was a RPE65 domain (residues 152-670) (Fig. 2). In addition, the
179
genomic DNA fragment of EcNinaB-X1 with the corresponding cDNA sequence was obtained,
180
which showed that it was composed of 12 exons and 11 introns (Fig.3). All intron-exon boundaries
181
are consistent with the consensus splicing junctions at both the 5’ splice donor site (GT) and the 3’
182
splice acceptor site (AG) of each intron.
183
A multiple sequence alignment showed that EcNinaB-X1 displayed high identities with
184
carotenoid isomerooxygenase of Armadillidium vulgare (AvNinaB-X1, 60%), and Nilaparvata
185
lugens (NlNinaB-X1, 51%) (Fig. 4).
186
Amino acid sequences of carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
187
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2) from different species
188
were collected from the NCBI database and a phylogenetic tree was constructed using
189
Neighbor-joining method. The phylogenetic analysis showed that EcNinaB-X1 was divided into
190
the same branch of NinaB (Fig.5).
191 192
3.2 Tissue distribution of EcNinaB-X1 mRNA
193
Expression profiling of EcNinaB-X1 mRNA in different tissues of E. carinicauda was
194
detected by quantitative real-time PCR. The expression of EcNinaB-X1 was expressed
195
constitutively in all of the detected tissues and highly expressed in the hepatopancreas, gill, and
196
stomach (Fig. 6).
197 198
3.3. Knockout of EcNinaB-X1 using CRISPR/Cas9 technology
199
Ten days after injection to the one-cell embryo, ten embryos were selected randomly in each
200
group and extracted the genomic DNA. Then the target fragment was amplified with detection
201
primers. The sequencing results of the purified PCR products showed that multiple peaks occurred
202
initially after the PAM sites compared with blank and control groups (Fig. 7). It indicated that the
203
genome of prawns in the experimental group had been successfully edited. In order to identify the
204
result, cloning and Sanger sequencing were carried out. Results showed that seven types of
205
deletion mutation were generated in total, but no insertions were found (Fig. 8).
206
The mutation rate of the embryos injected with Cas9 mRNA and one sgRNA was analyzed
207
and compared with the results of our previous research (Table 2). Of the 400 injected one-cell
208
stage embryos with Cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 58
209
embryos could develop to postlarvae and the reproductive survival rate was 14.5%. Fifty days
210
later, when the 58 lived embryos developed to adult E. carinicauda, one leg of them was collected
211
respectively to detect indels and the results indicated that the number of mutant prawns was 26
212
and the mutant rate reached 6.5%. Furtherly, of the 26 mutant prawns, 5 kinds of mutant types
213
were identified in the targeted locus (Fig. 9).
214 215
3.4. Evaluation of CRISPR/Cas9 generated EcNinaB-X1-KO prawns
216
To determine the effect of the EcNinaB-X1-KO on the prawns, the phenotypes and dissected
217
hepatopancreas of wild-type and EcNinaB-X1-KO prawns were compared. The hepatopancreas
218
from EcNinaB-X1-KO prawns exhibited a red color compared with wild-type individuals (Fig.
219
10A, B). According to the reference [27], the industrial color criteria was used to evaluated the
220
color change in the hepatopancreas between wild-type and EcNinaB-X1-KO prawns, and the
221
EcNinaB-X1-KO prawns displayed a significant shift to red color compared to the wild-type
222
groups (Fig. 10C).
223
The effects of EcNinaB-X1-KO on the mortality of bacteria-challenged prawns by
224
challenging them with were also evaluated. The prawns in EcNinaB-X1-KO group had
225
significantly low mortality than those in wild-type group when the prawns were challenged with V.
226
parahaemolyticus or A. hydrophila (Fig. 11A, B). For the Vibrio-challenged prawns, the mortality
227
reached 50% in wild-type group and only 20% in EcNinaB-X1-KO group at 48 h post-challenge
228
and there was significant difference between wild-type and EcNinaB-X1-KO prawns (p < 0.01).
229
For the Aeromonas-challenged prawns, the mortality reached 65% in wild-type group and only 25%
230
in EcNinaB-X1-KO group at 48 h post-challenge and there was significant difference between
231
wild-type and EcNinaB-X1-KO prawns (p < 0.05). The negative control showed a cumulative
232
mortality of ~10% indicating that PBS-injection itself was non-toxic in prawns. Overall, these
233
results indicated that the deletion of EcNinaB-X1 in E. carinicauda decreased prawn’s mortality
234
following V. parahaemolyticus or A. hydrophila challenge.
235 236
4. Discussion
237
Carotenoids are naturally occurring red, orange and yellow pigments that are synthesized by
238
plants, alga and some microorganisms and fulfill many important physiological functions [28].
239
Carotenoids are fat-soluble compounds, and it is important to enhance the bioavailability of
240
β-carotene from food or supplements in animals [29]. β-carotene is involved in a number of
241
beneficial functions in animals such as taking part in crucial signaling functions of its metabolites
242
and working as an antioxidant [27]. In addition, the accumulation of carotenoids in animals could
243
change the fat color in some animals, which make it important in animal breeding [27, 30]. Li et al
244
[31] cultured a new variety of Yesso scallop (Patinopecten yessoensis) with orange adductor
245
muscle, the ‘Haida golden scallop’, which was caused by carotenoid accumulation.
246
In animals, carotenoids could be cleaved into a range of biologically important products and
247
carotenoid oxygenases play an important role in the reaction [32]. It is reported that there are some
248
carotenoid oxygenases, including carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
249
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2) [20, 21]. In this
250
research, the full-length cDNA and genomic DNA of one carotenoid isomerooxygenase gene
251
(EcNinaB-X1) were obtained from E. carinicauda. There is one typical RPE65 domain at the
252
position of 152-670 in the deduced EcNinaB-X1 amino acid sequence. From the bioinformatic
253
analysis, we could find that the RPE65 domain played the oxidoreductase activity acting on single
254
donors with incorporation of molecular oxygen, or two atoms of oxygen [29]. Among three typical
255
carotenoid oxygenases, the phylogenetic analysis showed that EcNinaB-X1 was divided into the
256
same branch of NinaB, not belonging to β, β-carotene 15, 15'-monooxygenase (BCMO) and β,
257
β-carotene 9', 10'-oxygenase (BCO2).
258
Niu et al [27] reported that the biallelic modification of BCO2 from sheep resulted in yellow
259
fat, compared with the fat color in wild types (snow-flower white). Vage & Boman [33] reported
260
that a nonsense mutation in the BCO2 gene was tightly associated with accumulation of
261
carotenoids in adipose tissue of sheep (Ovis aries). Strychalski et al [34] reported that an
262
AAT-deletion mutation in the coding sequence of the BCO2 gene was related to the formation of
263
yellow-fat rabbit. Kyle-Little et al [35] reported that the mutant of BCMO1 gene affected macular
264
pigment optical density in young healthy caucasians. Amengual et al [36] reported that the
265
BCMO1 acted as a critical molecular player in deducing body adiposity of mice. However, there
266
was no report about the biological function of NinaB in regulating the physiology and
267
biochemistry of animals. In this research, we used CRISPR-Cas9 system to disrupt EcNinaB-X1
268
gene and obtained wild-type and homozygous mutants at EcNinaB-X1 loci in the offspring. In this
269
study, wild-type and homozygous EcNinaB-X1-KO prawns were selected as the experimental
270
animals to study the function of EcNinaB-X1 in immune defense. EcNinaB-X1-KO prawns
271
showed much lower mortality than those in wild-type group after V. parahaemolyticus or A.
272
hydrophila challenge, which indicated that EcNinaB-X1 might play a key role in immune defense
273
of prawns. In addition, the deletion of EcNinaB-X1 gene resulted in the color shift compared with
274
wild-type prawns. From above research, we concluded that NinaB-X1 gene could be used as a
275
candidate gene in marker-assistant selection in animal breeding.
276 277 278
Conflict of interest There is no conflict of interest.
279 280
Acknowledgments
281
This work was supported by the National Key R&D Program of China (No.
282
2018YFD0900205), National Natural Science Foundation of China (Grant Nos. 31872613,
283
41876196), The Natural Science Foundation of Hebei Province of China (Grant Nos.
284
C2019201236, D2019201239), Innovation & entrepreneurship training program for college
285
students of Hebei Province (Grant No. S201910075061), Innovation & entrepreneurship training
286
program for college students of Hebei University (Grant No. 2019177)， and Hangzhou Qianjiang
287
Special Expert for Jiquan Zhang. We are grateful to Dr. Huan Gao for providing the prawns.
288 289
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290 291 292 293 294 295 296 297 298 299 300 301 302 303
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Table 1 Primers mentioned in the paper
383
Primers
384 385
Sequences (5’- 3’)
Sequence information
EcNinaF
ACAGCCCCTTGTCGTGTCCGT
Real-time PCR
EcNinaR
TCGTCGCTCCCGTGTCCCT
Real-time PCR
18S-F
TATACGCTAGTGGAGCTGGAA
Real-time PCR
18S-R
GGGGAGGTAGTGACGAAAAAT
Real-time PCR
DetEcNinaF
TTTCAGTAACTCTTACGATTC
Detection of mutation
DetEcNinaR
CCCGCAGTGGTAGGAATAGG
Detection of mutation
Note: F and R stand for forward primers and reverse ones, respectively.
Table 2 Mutation frequencies induced by microinjection of Cas9 mRNA and EcNinaB-X1 gRNA
386
Injected Group
Survival
Mutant
Survival
Mutant
RNA concentration
100 ng/µL
Embryos
Postlarvae
Postlarvae
Rate
Rate
247
35
18
14.17%
7.29%
[11]
250
23
12
9.20%
4.80%
[9]
400
58
26
14.50%
6.50%
In this paper
200 ng/µL
EcChi4 (gRNA-EcChi4)
(pCMV-Cas9)
100 ng/µL
200 ng/µL
EcMIH (gRNA-EcMIH)
(pCMV-Cas9)
100 ng/µL
200 ng/µL
EcNinaB-X1 (gRNA-EcNinaB-X1)
References
(pCMV-Cas9)
387
Legends of Figures
388
Fig.1 Nucleotide and deduced amino acid sequences of EcNinaB-X1 gene.
389
Nucleotides are numbered on the both sides of the sequence. The letters marked with double
390
underline represented the RPE65 domains.
391
Fig.2 The domain architecture of deduced EcNinaB-X1.
392
Fig. 3 Schematic representation of the structure of EcNinaB-X1 gene depicting exons, gRNA
393
target sequence (sequence in blue at the bottom) and PAM site (in red). The numbers indicate the
394
exact lengths of the exons.
395
Fig. 4 Alignment of the amino acid sequence of EcNinaB-X1 with other known NinaBs.
396
The identical residues are shown in solid boxes. Sequences start at the first methionine residue.
397
Armadillidium vulgare (AvNinaB-X1, GenBank accession no. RXG54508.1); Nilaparvata lugens
398
(NlNinaB-X1, XP_022185479.1); E. carinicauda (EcNinaB-X1, MN583041, in this research).
399
Fig.5 Phylogenetic tree of carotenoid isomerooxygenase (NinaB), β, β-carotene 15,
400
15'-monooxygenase (BCMO) and β, β-carotene 9', 10'-oxygenase (BCO2) from different species
401
based on the amino acid sequence comparisons.
402
Aedes albopictus (AaNinaB-X1, GenBank accession no. XP_019545564.1); Aethina tumida
403
(AtNinaB-X1, XP_019870879.1); Anoplophora glabripennis (AgNinaB-X1, XP_023310515.1);
404
Apis cerana (AcNinaB-X1, XP_016913671.1); A. dorsata (AdNinaB-X1, XP_006611100.1); A.
405
florea
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Armadillidium vulgare (AvNinaB-X1, GenBank accession no. RXG54508.1); Bombus terrestris
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(BtNinaB-X1, XP_003400564.1); Branchiostoma belcheri (BbBCO2-X1, XP_019636508.1);
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Callorhinchus milii (CmBCMO-X1, XP_007887463.1); Ceratina calcarata (CcNinaB-X1,
(AfNinaB-X1,
XP_003695005.1);
A.
mellifera
(AmNinaB-X1,
XP_394000.4);
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XP_017875448.1); Clupea harengus (ChBCO2-X1, XP_012685829.1); Dendroctonus ponderosae
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(DpNinaB-X1, XP_019773593.1); Diachasma alloeum (DaNinaB-X1, XP_015113077.1);
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Dinoponera quadriceps (DqNinaB-X1, XP_014471901.1); Dufourea novaeangliae (DnNinaB-X1,
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XP_015439200.1); Eufriesea mexicana (EmNinaB-X1, XP_017759905.1); Fopius arisanus
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(FaNinaB-X1, XP_011310428.1); Fundulus heteroclitus (FhBCMO-X1, XP_012709399.1);
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Habropoda laboriosa (HlNinaB-X1, XP_017798623.1); Harpegnathos saltator (HsNinaB-X1,
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XP_011149992.1); Ictalurus punctatus (IpBCO2-X1, XP_017316112.1); Latimeria chalumnae
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(LcBCMO-X1,
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XP_014345540.1;
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XP_013396242.1); Megachile rotundata (MrNinaB-X1, XP_003702796.1); Nilaparvata lugens
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(NlNinaB-X1, XP_022185479.1); Pseudomyrmex gracilis (PgNinaB-X1, XP_020278007.1);
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Salmo salar (SsBCMO-X1, XP_014030705.1; SsBCMO-X2, XP_014030708.1; SsBCMO-X3,
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XP_014030708.1); Sarcophilus harrisii (ShBCMO-X1, XP_003757903.1); Trichogramma
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pretiosum (TpNinaB-X1, XP_014224940.1); E. carinicauda (EcNinaB-X1, MN583041, in this
423
research). Values on the line are bootstrap values showing percentage confidence of relatedness.
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Fig.6 Detection of EcNinaB-X1 transcripts in different tissues of E. carinicauda. Tissues were
425
shown in the abscissa. The amount of EcNinaB-X1 mRNA was normalized to the 18S rRNA
426
transcript level. Data are shown as means ± SD (standard deviation) of three separate individuals
427
in the tissues.
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Fig.7 Sanger sequencing of the PCR products from 10 prawns indicated the indel mutations
429
caused by CRISPR/Cas9 genome editing system. WT means the wild-type prawns (blank group).
430
M1 and M2 mean injected embryos. The PAM site was represented in pink rectangles.
XP_006004911.1; LcBCO2-X3,
LcBCO2-X1, XP_014345541.1);
XP_014345536.1; Lingula
anatina
LcBCO2-X2, (LaBCO2-X1,
431
Fig.8 Sequencing of CRISPR/Cas9 treated prawns. The wild-type sequence (ref) is shown at the
432
top. The gRNA site is underlined and the sequence in pink frame (TGG) represents the PAM
433
sequence. The numbers on the right represents the number of mutations recovered by sequencing.
434
Within the sequences, deletions are indicated by dashed lines.
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Fig. 9 Sanger sequencing of the PCR products from prawns individually indicated the indel
436
mutations caused by CRISPR/Cas9 genome editing system. WT means the wild-type prawn (blank
437
group). MT-1, 2, 3, 4, and 5 mean five mutant-type prawns. The PAM site was represented in
438
black rectangles.
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Figure 10 Phenotypes and dissection of EcNinaB-X1 knockouts in the hepatopancreas of E.
440
carinicauda. (a) Phenotypes of wild-type and EcNinaB-X1-KO prawns. (b) Hepatopancreas
441
dissected from prawns showed in figure (a). (c) Color differences in the hepatopancreas of
442
wild-type and EcNinaB-X1-KO prawns; ‘R’ indicates red color, ‘G’ indicates green color, ‘B’
443
indicates blue color. According to the reference [27], we defined the (R+G)/B value to indicate the
444
red color.
445
Fig. 11 The mortality of the prawns in wild-type group and EcNinaB-X1-KO group after the
446
prawns were challenged with Vibrio parahaemolyticus and equal volume of PBS (A), or
447
Aeromonas hydrophila and equal volume of PBS (B) at 0, 12, 24, 48, 72, 96, and 120 h.
448
449
450 Fig.1
451 452 453
.
454 455 456
Fig.2
457 458 459 460
Fig. 3
461 462 463
Fig. 4
464 465 466
Fig.5
467 468 469
Fig.6
470
471 472 473
Fig.7
474 475 476
Fig. 8
477
478 479 480
Fig. 9
481
482
(a)
483
(b)
(c)
484 485
Figure 10
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.
487
488 489
Fig. 11
Highlights
EcNinaB-X1 gene was cloned from E. carinicauda. The expression profiles of EcNinaB-X1 were demonstrated. CRISPR-Cas9 system efficiently generated indels in EcNinaB-X1 loci. EcNinaB-X1-KO prawns had lower mortality than wild-type after bacterial challenge.