mediated knockout of the gene in the global

The diamondback moth, Plutella xylostella (L.), is a worldwide agricultural pest that has developed resistance to multiple classes of insecticides. Genetics-based approaches show promise as alternative pest management approaches but require functional studies to identify suitable gene targets. Here we use the CRISPR/Cas9 system to target a gene, abdominal-A , which has an important role in determining the identity and functionality of abdominal segments. We report that P . xylostella abdominal - A ( Pxabd - A ) has two structurally-similar splice isoforms (A and B) that differ only in the length of exon II, with 15 additional nucleotides in isoform A. Pxabd-A transcripts were detected in all developmental stages, and particularly in pupae and adults. CRISPR/Cas9-based mutagenesis of Pxabd-A exon I produced 91% chimeric mutants following injection of 448 eggs. Phenotypes with abnormal prolegs and malformed segments were visible in hatched larvae and unhatched embryos, and various defects were inherited by the next generation (G 1 ). Genotyping of mutants demonstrated several mutations at the Pxabd-A genomic locus. The results indicate that a series of insertions and deletions were induced in the Pxabd-A locus, not only in G 0 survivors but also in G 1 individuals, and this provides a foundation for genome editing. Our study demonstrates the utility of the CRISPR/Cas9 system for targeting genes in an agricultural pest and therefore provides a foundation the development of novel pest management tools. © 2016 Elsevier Ltd. All rights reserved.


Introduction
The diamondback moth (DBM), Plutella xylostella (L.), is one of the most destructive and cosmopolitan pests of cruciferous crops. It attacks many economically important food crops such as oilseed rape and cabbage, and the annual total cost of damage and management worldwide is estimated at USD4-5 billion (Furlong et al., 2013;Zalucki et al., 2012). Plutella xylostella has developed resistance to all major classes of pesticides, including dichlorodiphenyl-trichloroethane (DTT) and Bacillus thuringiensis (Bt) (Angkersmit, 1953;Johnson, 1953;Tabashnik et al., 1990), making it difficult to control and demanding the development of novel management strategies. Genetics-based technologies such as pig-gyBac-mediated transgenesis, support the ability to develop population-suppression DBM strains by release of insects carrying a dominant lethal trait (RIDL) (Martins et al., 2012). Although pig-gyBac is a versatile transposon element for engineering insects, its random integration, relatively low transforming frequency, possible instability of integrated sequences, and limited carrying capacity hamper its wide application in pest control trials (Fraser Jr, 2012).
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein) is a newly emerged genomeediting tool with advantages over the earlier, time-and laborconsuming techniques: zinc finger nucleases (ZFN) and transcription activator-like effecter nucleases (TALENs) (Miller et al., 2007Porteus and Baltimore, 2003;Sander et al., 2011;Wood et al., 2011). This novel methodology needs only the Cas9 nuclease coupled with a single guide RNA (sgRNA) to cleave a specific sequence. Since the first demonstration of engineering the type II CRISPR-Cas9 system to function in eukaryotes (Cong et al., 2013), the technique has been used to edit genomes of numerous species including human (cells), mice, nematodes (Caenorhabditis elegans) and insects (Drosophila melanogaster), and for substantially improving the ease of genome editing and studies of genome regulation (Cho et al., 2013;Friedland et al., 2013;Gilbert et al., 2013;Gratz et al., 2013;Li et al., 2013;Qi et al., 2013).
The abdominal-A (abd-A) gene, belonging to the homeotic gene (Hox) family, is a member of Drosophila bithorax complex, which is required for segmental identity during embryogenesis (S anchez-Herrero et al., 1984). These genes encode transcription factors that modulate segment development by interacting with a large number of downstream target genes (Pavlopoulos and Akam, 2011). Gene products of abd-A are involved in many biological processes during early embryogenesis of Drosophila and other insects. These include the differentiation of the anterior body and rear somite axis, cardiac tube organogenesis, heart cell fate in the dorsal vessel, genesis of the nervous system and fat body, gonad formation and development, midgut formation and muscle patterning (Cumberledge et al., 1992;Foronda et al., 2006;Lovato et al., 2002;Marchetti et al., 2003;Mathies et al., 1994;Michelson, 1994;Perrin et al., 2004;Ponzielli et al., 2002). Products of abd-A also play a role in larva-to-pupa metamorphosis in the silkworm, Bombyx mori, and abdominal pigmentation of adult D. melanogaster (Deng et al., 2012;Rogers et al., 2014). RNAi of abd-A in silkworms results in complete or partial absence of ventral appendages (prolegs and legs) from the third to sixth abdominal segments in late-stage embryos, indicating its importance in the normal development of these segments (Pan et al., 2009).
We cloned the P. xylostella ortholog, designated Pxabd-A, profiled levels of expression across different life stages and sexes, and applied the CRISPR/Cas9 system to generate loss-of-function individuals and visible defect phenotypes. Severe abdominal morphological defects and significant lethality resulted from disruption of the gene. Our results demonstrate the possibility of further gene function studies based on genome editing and developing novel approaches for genetic control of this globally important pest insect.

Experimental DBM strain
The experimental DBM strain (Fuzhou-S) was derived from insecticide-susceptible insects collected from a cabbage (Brassica oleracea var. capitata) crop in Fuzhou (26.08 N,119.28 E) in 2004 and later used for genome sequencing . Larvae were reared on potted radish seedlings (Raphanus sativus L.) at 25 ± 1 C, 65 ± 5%RH and L:D ¼ 16:8 h, in a greenhouse without exposure to insecticides.

Cloning of Pxabd-A
Total RNA was isolated from five P. xylostella 3 rd instar larvae using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized with Hiscript™ Reverse Transcriptase (Vazyme Biotech) by using 500 ng total RNA. The Pxabd-A cDNA was amplified with a pair of primers (F: 5 0 -ATG AGT TCC AAG TTC ATC ATC G -3 0 ; R: 5 0 -TTA CGT GGG CAC CTT GTT GA -3 0 ) corresponding to the predicted coding sequence of Pxabd-A (gene ID in DBM genome: Px004264) in the P. xylostella genome (http:// iae.fafu.edu.cn/DBM/index.php). PCR was carried out with KODplus polymerase (TOYOBO, Japan) under the following conditions: 98 C for 2 min, and then 30 cycles at 98 C for 30 s, 55 C for 30 s, 68 C for 70 s, and a final elongation step at 68 C for 10 min. PCR products were isolated on a 1% agarose gel stained with ethidium bromide. The target band was extracted using the Omega gel extraction kit (Omega) and cloned into PJET1.2 vector (Thermo scientific) for sequencing.

Alignment and phylogenetic analysis of Pxabd-A
Multiple alignments were conducted using the MUSCLE algorithm based on the amino acid sequences encode by abd-A orthologs from 12 species (P. xylostella; B. mori, Bombus terrestris, Apis mellifera, Tribolium castaneum, Anopheles gambiae, Drosophila melanogaster, Acyrthosiphon pisum, Myrmica rubra, Strigamia maritima, Hymenolepis microstoma and Echinococcus granulosus) available in GenBank. The phylogenetic tree was constructed using the maximum likelihood method with a bootstrap value of 1,000 in MEGA5.1.

In vitro transcription of Cas9 and sgRNA
A 23 base-pair (bp) sgRNA targeting site located at nucleotides 163e185 bp (5 0 -GGA CTG AGT GCA GCG GCT CTA GG -3 0 ) was selected in Exon I of Pxabd-A. The control sgRNA (5 0 -GGC GAG GGC GAT GCC ACC TA -3 0 ) was used to target the exogenous gene encoding the EGFP protein, and the targeted efficiency of EGFP-sgRNA verified in a DBM embryo cell line (established in our laboratory). The sgRNA was generated from a ready-to-use 500 bp linearized vector by annealing oligonucleotide duplexes encoding the 20 bp target sequence upstream of protospacer adjacent motif (PAM), with the sgRNA expressed under control of the T7 promoter. The sgRNA template was subcloned into the PJET1.2 vector (Fermentas, Ontario, CA) and sequenced to verify the structure. sgRNA was synthesized in vitro using the MAXIcript T7 kit (Ambion). The PTD1-T7-Cas9 vector (ViewSolid Biotech, Beijing, China) was linearized with the NotI restriction enzyme (Fermentas, US), and the Cas9 mRNA synthesized in vitro using the mMESSAGE mMACHINE T7 Kit (Ambion).

Embryo microinjection
Female P. xylostella adults were caged to allow oviposition on 10 cm 2 parafilm sheets treated previously with a cabbage leaf extract. The sheets were renewed at intervals of 30 min to obtain fresh eggs. Eggs were aligned individually along the same anteriorposterior axis (Fig. S1) and microinjected with 300 ng/ml or 500 ng/ ml of Cas9 mRNA along with 150 ng/ml sgRNA into the posterior pole of each egg within one hour of oviposition. Eggs were incubated at 25 C.

Phenotype screening
Injected eggs were checked for development to calculate the hatching rate, and observed under a stereo microscope (Nikon, SW-2B/22) to examine the abdominal morphology of embryos on the third day after injection. Hatched larvae were observed under a microscope to detect mutated G 0 individuals that were subsequently reared on radish plants at 25 C. The mutant phenotypes were imaged digitally with a stereo microscope.

T7 endonuclease I assay and characterization of targeted mutant loci
To confirm the mutagenesis of the Pxabd-A locus, genomic DNA from the 4 th instar somatic mutants or wild-types was extracted individually (DNA extraction kit; CWBio, China). PCR was performed to amplify the fragments surrounding the sgRNA targeted sites from the genomic DNA samples using Phusion High-Fidelity DNA polymerase (New England Biolabs [NEB]) with Pxabd-A target site-specific primers (F: 5 0 -GCA GTT CCA TCA TCA GAA CTT G -3 0 ; R: 5 0 -CAG TGA TGG ACA TCC AAG GAT A -3 0 ). A hybridization reaction was run with 19 ml mixtures containing 200 ng of the PCR products, 2 ml NEB buffer 2 and ddH 2 O in a PCR cycler: 95 C for 5 min; ramping down with À2 C/s to 85 C and with À0.1 C/s to 25 C; and finally holding at 4 C. The mixture then was treated with 1 ml of T7 endonuclease I (NEB) at 37 C for 15 min and the reaction stopped by adding 2 ml of 0.25 M EDTA. The reaction mixture was resolved on a 2% agarose gel, and the DNA fragments analyzed under UV. The intensity of the fragments was quantified by Quantity one software (Bio-Rad, Hercules, California). PCR products of the mutants were ligated into pJET-Blunt vector and sequenced for validation of the genomic mutated events. Genomic DNA was prepared from 20 unhatched eggs to identify somatic mutations. The Gbdirect PCR kit (Genebank Biosciences Inc., China) was used to amplify potential mutated fragments. Amplified fragments were cloned into PJET1.2 (Fermentas, US) and sequenced.

Identification and characterization of Pxabd-A
The B. mori abd-A ortholog, Bmabd-A (GeneBank EU365399), was used as a query with blastn to identify the putative coding sequence of Pxabd-A from the P. xylostella genome . Pxabd-A was mapped to region 991,463e1,151,462 bp in scaffold 17 of the genome. Analysis of the complete coding sequence showed that Pxabd-A has two splice isoforms, which we designate A and B (Fig. 1A). Pxabd-A isoform A contains three exons (604, 60, and 401 bp in length) and two introns (4,977 and 23,213 bp), for a total coding sequence of 1,065 bp. Isoform B differs from isoform A in the length of exon II (45 bp) and the first intron (4,992 bp), and has a coding sequence 1,050 bp in length. Our abd-A phylogenetic tree shows a clear pattern of order-specific clustering within the Insecta, supporting the conclusion that the corresponding genes are conserved within each of the groups (Fig. 1B). Plutella xylostella and B. mori are clustered into the Lepidoptera clade and are divergent from the Diptera clade represented by the mosquito, Anopheles gambiae, and fruit fly, D. melanogaster. The isoforms are wellclustered in the insects (P. xylostella, B. mori, B. terrestris and D. melanogaster), supporting the conclusion that they result from a function-associated divergence within each species.

Expression profiling of Pxabd-A
Previous studies have established that abd-A transcripts are expressed in almost all abdominal segments during embryo development (Busturia et al., 1989;Peifer et al., 1987;S anchez-Herrero et al., 1984). However, in later developmental processes, abd-A was acting downstream of the genes that define position and cell type within segments, as intermediates in the hierarchy of control that leads to morphogenesis (Foronda et al., 2006;LaBeau et al., 2009;Peifer et al., 1987). qRT-PCR-based expression profiling showed that total Pxabd-A (isoforms A and B), and isoform A transcript accumulation levels were lower in 1 st -4 th instar larvae and prepupae compared with the egg, pupa, and adult (Fig. 2). Eggs and adult female and male insects accumulated the transcripts to levels 10-fold higher than the larval stages. The highest abundance, 15-fold greater than larval instars, was seen in female and male pupae. Isoform A exhibited the same level of transcript abundance between male/female pupae and adults. Pxabd-A transcripts accumulated were lower in female adults than male adults. Accumulation levels of isoform B transcripts were generally similar to isoform A, but higher in male adults compared with females (Fig. 2). Our finding that Pxabd-A was expressed in all developmental stages tested and both sexes provides a basis for further functional studies.

Phenotypes induced by disruption of Pxabd-A
To test the role of Pxabd-A in P. xylostella, a single target site in exon I of the Pxabd-A locus was selected for CRISPR/Cas9 mutagenesis (Fig. 4A). We injected Pxabd-A-sgRNA or EGFP-sgRNA (control) along with Cas9 mRNA into preblastoderm embryos. In the groups treated with Pxabd-A sgRNA, loss-of-function mutations of Pxabd-A were induced with a higher efficiency (91%) when injected with 500 ng/ml Cas9 mRNA when compared with injections of 300 ng/ml (35%) ( Table 1). Injection of structurally unrelated sgRNA for the EGFP control did not affect development of embryos and larvae. Hatchability of eggs was relatively low in the Pxabd-A specific sgRNA treatment. We checked individual unhatched eggs to determine if there was a visible phenotypic effect of Pxabd-A sgRNA. Some of the eggs developed sufficiently to form the black head capsule indicative of larvae (Fig. S2A), while 82 individual unhatched embryos exhibited a severe abdominal distortion (Fig. S2B). Most unhatched eggs from the EGFP-sgRNA injected control exhibited yellow coloration, possibly because of microinjection damage. The developing embryos with black head capsules had normal segmentation and prolegs. A total of 218 pupae (90%) developed and produced adults in the EGFP-sgRNA control, a rate similar to that reported for the wild-type (Peng et al., 2015).
In the Pxabd-A-sgRNA treatment, excluding the 82 mutated unhatched eggs, 281 of the 501 injected eggs hatched and produced larvae with mutant phenotypes in which abdominal segments were fused ( Fig. 3A and B) and 189 of these died within two days. A total of 92 larvae with less-severe mutations survived to the 4 th instar stage (Fig. 3C) and 73 of these pupated (Fig. 3D). Insects resulting from the CRISPR targeted Pxabd-A locus could be divided into two classes based on loss-of-function phenotypes. The first class had larval phenotypes with fused abdominal segments (Fig 3B), crochets absent from some prolegs (Fig. 3E) or deformed testis (Fig. 3F). The larvae with segment defects could walk only with their thoracic legs (Movie 1), and died within two days. The second class comprised adult phenotypes with external genitalia deviating from the central axis (Fig. 4A) and deformed testis (Fig. 4B). Of the 23 males, 21 (91%) were sterile. In the mutated G 0 , 57% of the female adults layed eggs. Sterile females that layed no eggs despite the presence of eggs in the ovary may have resulted from normal egg migration being disrupted as reported for the phenotypes from different mutations of abd-A in Drosophila (Cumberledge et al., 1992). It appears that sterility is caused, at least in part, by the disruption of gonad development induced by CRISPR/Cas targeting of Pxabd-A locus. Collectively these two classes of phenotypes support the interpretation Pxabd-A may play a largely permissive role in promoting segmentation and gonad development in the Fig. 2. qRT-PCR-based expression of Pxabd-A at different developmental stages and sexes of the adult. Abbreviations: E, eggs; L1, L2 represent 1 st , 2 nd instar larvae; L3M, L3F, L4M and L4F represent 3 rd and 4 th male/female instar larvae, respectively; PPM and PPF represent male and female prepupae; PM and PF represent male and female pupae; M and F represent male and female adults. Error bars indicate standard errors of the mean. Statistically-significant differences were labeled with different letters or letters in parentheses as analyzed with one-way ANOVA (Duncan's multiple range test, P < 0.05, n ¼ 3). diamondback moth.

CRISPR/Cas9 mediated mutagenesis of Pxabd-A
Genomic DNA from unhatched egg was used as a template to amplify the sgRNA target region and confirm that unhatched mutated eggs were caused by mutagenesis of Pxabd-A locus. Four of six sequenced clones (66%) were found to have insertion and deletion mutations caused by CRISPR/Cas9-induced nonhomologous end joining (Fig. 5C a). Furthermore, mutation events at the targeted region of the Pxabd-A locus in five randomly-selected, mutated 4 th instar larvae were examined by the T7EI assay, and the indel frequency ranged from~20% to~35%. All five mutants exhibited sharp and clear cleavage products (Fig. 5B), indicative of mutagenesis at the targeted locus. Sequencing results revealed that six of 15 clones (40%) had indel mutations from one 4 th instar larva (Fig. 5C, b). Consequently, the CRISPR/Cas9 system effectively induced mutagenesis at the Pxabd-A locus in the genome.

Germline transformation mediated by CRISPR/Cas9
A total of 23 somatic mutated males and 51 females, survived to the adult stage. Each of these mutants was outcrossed to wild-type a For each concentration, Cas9 mRNA with different sgRNAs (abd-A-sgRNA or EGFP-sgRNA) was injected into embryos. G 0 mosaic records the abnormal hatched larvae and dissected disorder unhatched eggs. The G 0 mutation rate ¼ (mutated larvae þ mutated unhatched eggs)/(hatched larvae þ mutated unhatched eggs). The germline transmission rate ¼ positive G 1 egg batches/G 1 egg batches. Each G 1 egg batch was from a different mutated G 0 parent (females and males). The positive G 1 egg batch indicates one and more offspring showing mutated phenotype and genotype. The germline transmission rate indicates the fraction of parents that produced one or more mutants in their germline. adults. The genetically transformed mutants among generation 1 (G 1 ) larvae were easily distinguishable based on the disorder of abdominal segments (Fig. 6A). Seven abnormal larvae were obtained following screening of 2,860 G 1 larvae from 33 egg batches. One abnormal G 1 larva exhibited hardening in the A4-A8 abdominal segments and loss of abdominal mobility (Movie. 2). Six G 1 mutated larvae exhibited phenotypes similar to those of G 0 larvae (Fig. 6B). All G 1 eggs layed by the wild-type females that had mated with mutated G 0 males desiccated and did not hatch (Fig. 6C), consistent with them being unfertilized. To verify the mutagenesis of Pxabd-A locus in G 1 larvae, we used DNA from a single individual as a template to perform direct PCR. Amplified fragments were  cloned into vector and sequenced. Sequencing results showed that the mutated allele of G 1 mutations exhibited deletion and insertion surrounding the targeted region of the Pxabd-A locus (Fig. 6D). The germline transmission efficiency was calculated by screening 33 egg batches with three positive egg batches (9.1%) that has at least one larva showing mutated phenotype and genotype (Table 1).

Discussion
We show here that the CRISPR/Cas9 system can be used for targeting genes in an agricultural pest. This system is demonstrated to induce a series of insertions and deletions in the P. xylostella abd-A. Our results also showed stable germline mutations mediated by the CRISPR/Cas9 system. The germline transmission rate was 9.1%, which is much lower than that described for CRISPR/Cas9 use in silkworm (35.6%) However the G 0 mutant rate (91%) is consistent with that seen in the silkworm (94%e100%) . The apparent deficit in DBM germline transmission efficiency may be attributable to the high percentage of sterility in males and females caused by loss-of-function of Pxabd-A. These results demonstrate that the Cas9/sgRNA can induce the intended mutagenesis in P. xylostella, and be used as a powerful tool for genome manipulation of this globally significant pest.
We chose the abd-A as a targeted gene since it is required for determining the correct identity of A2-A8 in Drosophila. A single mutated allele of abd-A disrupts gene expression causing a strong transformation of A2-A3 and a weaker transformation of A5-A7 towards A1; and mutations are homozygous embryonic lethal (Busturia et al., 1989;Karch et al., 1985). Disruption of Pxabd-A causes severe mutations: fusion and disorder of abdominal segments A3-A8 were observed in mosaic mutated DBM larvae, unlike the segment transformation reported in Drosophila (Busturia et al., 1989;Karch et al., 1985;S anchez-Herrero et al., 1984). The abd-A gene also is required for proleg development in the silkworm and they are missing following RNAi-mediated ablation of Bmabd-A transcripts (Pan et al., 2009). In contrast, our data showed proleg abnormalities resulting from mutations in Pxabd-A. In the adult stage, the external and internal genitalia of mutated males were abnormal. Previous reports in Drosophila showed that gonad formation and development require the abd-A domain, especially in the somatic cells of the gonad (Cumberledge et al., 1992;Foronda et al., 2006). This may explain the sterile females recovered in our study. Clearly, the detailed mechanisms resulting in the defects observed following mutating or ablating abd-A products require further analysis.
The abd-A genes play roles other than defining abdominal segment identities as evidenced by previous studies. It is common that most transcription factors (such as abd-A) serve multiple roles during development in insects. Therefore, the molecular mechanisms disrupted by Pxabd-A mutagenesis are too complicated to be completely revealed based on our present study. However, mutations of Pxabd-A were transmissible to G 1 progeny indicating the feasibility of the CRISPR/Cas9 system in non-model organisms. CRISPR/Cas9 mediated genome editing for P. xylostella gene function studies is still challenging because most genes are recessive so only homozygous mutants display phenotypes. Recently, the mutagenic chain reaction (MCR) technique based on CRISPR/Cas9  was shown to be efficient in converting heterozygous recessive mutations to a homozygous state and revealing mutant phenotypes. Furthermore, the method can be used to obtain transgenic insects carrying effecter genes, which can be expanded in field populations for pest management . P. xylostella is a worldwide agricultural pest that has developed resistance to multiple classes of insecticides. The simplicity and adaptability of CRISPR/Cas9 opens the door for revealing gene function and new avenues for management of this pest.