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RESEARCH REPORT TECHNIQUES AND RESOURCES Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum Anna F. Gilles 1,2, *, Johannes B. Schinko 1 and Michalis Averof 1,3, * ABSTRACT Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs. KEY WORDS: Gene editing, Insect, Evo-devo INTRODUCTION Until recently, gene targeting was a privilege of the few; it was possible only in a small number of organisms and involved sophisticated, labor-intensive techniques. Gene targeting in mammals required first modifying an allele in embryonic stem cells, then selecting the few cells carrying the targeting event and transplanting them into blastocysts to generate chimeras in which these cells would hopefully populate the germ line and contribute to the next generation (Capecchi, 2005; Smithies et al., 1985; Thomas et al., 1986). In Drosophila, after several failed efforts, efficient gene targeting was developed using a method that required bringing together three different transgenes (Rong and Golic, 2000). These techniques were not applicable to most other organisms, in which cultured pluripotent cells and sophisticated genetics were unavailable. The invention of zinc-finger and transcription activator-like effector (TALE) nucleases marked a big step in our ability to target genes efficiently, by directing double-strand breaks to chosen sites in the genome and exploiting the cellsendogenous DNA repair mechanisms to introduce changes at these sites (Carroll, 2014; Kim et al., 1996). Double-strand breaks are most frequently corrected by non-homologous end-joining (NHEJ) repair mechanisms, which can introduce small insertions or deletions (indels) at the site of repair. Less frequently, breaks are repaired through copying of a template that bears homologous sequences; such homology- directed repair (HDR) provides an opportunity to introduce specific changes into the locus via an engineered template. Zinc- finger and TALE nucleases proved to be extremely efficient for generating knockout and knock-in alleles compared with conventional gene targeting approaches. Their widespread use was limited mostly by the effort (and cost) required to customize their targeting specificity. The recent discovery of CRISPR/Cas nucleases, whose sequence specificity is guided by simple base complementarity between the target DNA and a small guide RNA (gRNA), provided a simple, efficient and affordable way of customizing nuclease specificity (Jinek et al., 2012; Sander and Joung, 2014). CRISPR/Cas nucleases consist of protein and RNA. Their specificity is determined by base complementarity with the 5end of the gRNA followed by the presence of a protospacer adjacent motif(PAM) in the target sequence. In the most commonly used CRISPR system, derived from Streptococcus pyogenes, the PAM sequence is NGG. Thus, by cloning the appropriate targeting sequence (N 17-20 ) at the 5end of a gRNA, it is possible to generate nucleases targeting any sequence that conforms to N 17-20 NGG (where N can be any nucleotide). Owing to this straightforward way of generating nucleases with a chosen sequence specificity and to its high targeting efficiency in a range of organisms, CRISPR technology holds great promise for emerging model organisms (Gilles and Averof, 2014). In principle, CRISPR-mediated gene targeting should be applicable to all organisms. In practice, the effectiveness of this approach is constrained by our ability to deliver CRISPR/Cas nucleases to cells of interest (e.g. to the germ line), by the nature and efficiency of the organisms DNA repair mechanisms and by our ability to identify and maintain the resulting mutants. These parameters will ultimately determine the feasibility and efficiency of gene targeting in a given species. Here, we present our effort to apply CRISPR technology in the beetle Tribolium castaneum and to establish methods and tools for efficient gene targeting in this species. Apart from being an important pest, infesting stored grain and grain products, Tribolium castaneum has emerged as an attractive experimental model for comparative developmental biology. Starting with classic genetic Received 7 April 2015; Accepted 29 June 2015 1 Institut de Gé nomique Fonctionnelle de Lyon (IGFL), École Normale Supé rieure de Lyon, 46 Allé edItalie, Lyon 69264, France. 2 École doctorale BMIC, Université Claude Bernard, Lyon 1, France. 3 Centre National de la Recherche Scientifique (CNRS), France. *Authors for correspondence ([email protected]; [email protected]) 2832 © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2832-2839 doi:10.1242/dev.125054 DEVELOPMENT

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RESEARCH REPORT TECHNIQUES AND RESOURCES

Efficient CRISPR-mediated gene targeting and transgenereplacement in the beetle Tribolium castaneumAnna F. Gilles1,2,*, Johannes B. Schinko1 and Michalis Averof1,3,*

ABSTRACTGene-editing techniques are revolutionizing the way we conductgenetics in many organisms. The CRISPR/Cas nuclease hasemerged as a highly versatile, efficient and affordable tool fortargeting chosen sites in the genome. Beyond its applications inestablished model organisms, CRISPR technology provides aplatform for genetic intervention in a wide range of species, limitedonly by our ability to deliver it to cells and to select mutationsefficiently. Here, we test the CRISPR technology in an emerginginsect model and pest, the beetle Tribolium castaneum. We usesimple assays to test CRISPR/Cas activity, we demonstrate efficientexpression of guide RNAs and Cas9 from Tribolium U6 and hsp68promoters and we test the efficiency of knockout and knock-inapproaches in Tribolium. We find that 55-80% of injected individualscarry mutations (indels) generated by non-homologous end joining,including mosaic bi-allelic knockouts; 71-100% carry such mutationsin their germ line and transmit them to the next generation. We showthat CRISPR-mediated gene knockout of the Tribolium E-cadheringene causes defects in dorsal closure, which is consistent withRNAi-induced phenotypes. Homology-directed knock-in of markertransgenes was observed in 14% of injected individuals andtransmitted to the next generation by 6% of injected individuals.Previous work in Tribolium mapped a large number of transgeneinsertions associated with developmental phenotypes and enhancertraps. We present an efficient method for re-purposing theseinsertions, via CRISPR-mediated replacement of these transgenesby new constructs.

KEY WORDS: Gene editing, Insect, Evo-devo

INTRODUCTIONUntil recently, gene targeting was a privilege of the few; it waspossible only in a small number of organisms and involvedsophisticated, labor-intensive techniques. Gene targeting inmammals required first modifying an allele in embryonic stemcells, then selecting the few cells carrying the targeting event andtransplanting them into blastocysts to generate chimeras in whichthese cells would hopefully populate the germ line and contribute tothe next generation (Capecchi, 2005; Smithies et al., 1985; Thomaset al., 1986). In Drosophila, after several failed efforts, efficientgene targeting was developed using a method that required bringingtogether three different transgenes (Rong and Golic, 2000). Thesetechniques were not applicable to most other organisms, in which

cultured pluripotent cells and sophisticated genetics wereunavailable.

The invention of zinc-finger and transcription activator-likeeffector (TALE) nucleases marked a big step in our ability to targetgenes efficiently, by directing double-strand breaks to chosen sitesin the genome and exploiting the cells’ endogenous DNA repairmechanisms to introduce changes at these sites (Carroll, 2014; Kimet al., 1996). Double-strand breaks are most frequently corrected bynon-homologous end-joining (NHEJ) repair mechanisms, whichcan introduce small insertions or deletions (indels) at the site ofrepair. Less frequently, breaks are repaired through copying of atemplate that bears homologous sequences; such homology-directed repair (HDR) provides an opportunity to introducespecific changes into the locus via an engineered template. Zinc-finger and TALE nucleases proved to be extremely efficient forgenerating knockout and knock-in alleles compared withconventional gene targeting approaches. Their widespread usewas limited mostly by the effort (and cost) required to customizetheir targeting specificity.

The recent discovery of CRISPR/Cas nucleases, whose sequencespecificity is guided by simple base complementarity between thetarget DNA and a small guide RNA (gRNA), provided a simple,efficient and affordable way of customizing nuclease specificity(Jinek et al., 2012; Sander and Joung, 2014). CRISPR/Casnucleases consist of protein and RNA. Their specificity isdetermined by base complementarity with the 5′ end of thegRNA followed by the presence of a ‘protospacer adjacent motif’(PAM) in the target sequence. In the most commonly used CRISPRsystem, derived from Streptococcus pyogenes, the PAM sequence isNGG. Thus, by cloning the appropriate targeting sequence (N17-20)at the 5′ end of a gRNA, it is possible to generate nucleases targetingany sequence that conforms to N17-20NGG (where N can be anynucleotide).

Owing to this straightforward way of generating nucleases with achosen sequence specificity and to its high targeting efficiency in arange of organisms, CRISPR technology holds great promise foremerging model organisms (Gilles and Averof, 2014). In principle,CRISPR-mediated gene targeting should be applicable to allorganisms. In practice, the effectiveness of this approach isconstrained by our ability to deliver CRISPR/Cas nucleases tocells of interest (e.g. to the germ line), by the nature and efficiencyof the organism’s DNA repair mechanisms and by our ability toidentify and maintain the resulting mutants. These parameters willultimately determine the feasibility and efficiency of gene targetingin a given species.

Here, we present our effort to apply CRISPR technology in thebeetle Tribolium castaneum and to establish methods and tools forefficient gene targeting in this species. Apart from being animportant pest, infesting stored grain and grain products, Triboliumcastaneum has emerged as an attractive experimental model forcomparative developmental biology. Starting with classic geneticReceived 7 April 2015; Accepted 29 June 2015

1Institut deGenomique Fonctionnelle de Lyon (IGFL), École Normale Superieure deLyon, 46 Allee d’Italie, Lyon 69264, France. 2École doctorale BMIC, UniversiteClaude Bernard, Lyon 1, France. 3Centre National de la Recherche Scientifique(CNRS), France.

*Authors forcorrespondence ([email protected]; [email protected])

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screens conducted in the 1980s (Beeman et al., 1989; Sulston andAnderson, 1996), the Tribolium research community has grown,bringing a plethora of genetic tools and approaches to this species.These approaches include transgenesis (Berghammer et al., 1999,2009; Lorenzen et al., 2003; Pavlopoulos et al., 2004), RNAi-mediated knockdown (Bucher et al., 2002; Posnien et al., 2009),enhancer trapping (Lorenzen et al., 2003; Trauner et al., 2009), heat-shock and GAL4/UAS-mediated mis-expression (Schinko et al.,2012, 2010) and live imaging (Benton et al., 2013; Sarrazin et al.,2012; Strobl and Stelzer, 2014). Moreover, the Tribolium genome issequenced and well annotated (Kim et al., 2010; Richards et al.,2008). Based on these tools and resources, Tribolium castaneum hasbecome the most advanced genetically tractable insect model afterDrosophila.Establishing efficient gene targeting in Tribolium brings to this

model a more precise tool for reverse genetics, a directed approachfor generating new markers and GAL4 drivers, and the opportunityto generate balancer chromosomes (for a more comprehensivediscussion on the potential uses of CRISPR in emerging models, seeGilles and Averof, 2014).

RESULTS AND DISCUSSIONTargeting of a genomic EGFP insertion using CRISPRWe established an efficient assay for scoring gene targeting eventsin Tribolium, making use of the enhancer trap line Pig-19 (Lorenzenet al., 2003). Pig-19 is homozygous for a single insertion ofpBac(3xP3-EGFP) in the Tribolium genome. Transgenic animalshave strong enhanced green fluorescent protein (EGFP)

fluorescence in larval, pupal and adult muscles (Fig. 1A,B). As aproof of concept for the functionality of CRISPR/Cas9 inTribolium, we injected Pig-19 embryos with in vitro transcribedeGFP1 gRNA, targeting the EGFP coding sequence 116 bp afterthe start codon (Auer et al., 2014), together with capped Cas9mRNA.

Three days after injection, the freshly hatched larvae werescrutinized for loss of EGFP expression in muscles. EGFP wasvisibly altered in 70-80% of the larvae (Table 1, rows 1, 2). Themildest phenotypes were small patches of non-fluorescent muscles,the strongest were loss of EGFP fluorescence in muscles of allsegments (Fig. 1C). The loss of EGFP from muscles indicates bi-allelic targeting of EGFP in all the nuclei of the multinucleatedmuscle fiber, a sign of highly efficient somatic gene knockout.

To assess the frequency of germline targeting events, we raisedinjected (G0) animals and crossed them out to adult vermilionwhite

beetles (Lorenzen et al., 2002). We then screened the progeny ofeach cross (G1) and determined the number of G0 animals that gaverise to non-fluorescent progeny (founders). Seventy-one percent offertile crosses produced non-fluorescent progeny (Table 1, row 2).G0 animals showing mosaic loss of EGFP were more likely toproduce non-fluorescent offspring (21 out of 22) compared with G0animals with normal EGFP fluorescence (6 out of 16), suggestingthat somatic targeting in G0 larvae may be a useful indicator ofgermline targeting in these animals.

The proportion of non-fluorescent larvae emerging fromeach cross was variable, ranging from 8% to 100% of G1animals. Sequencing of the EGFP transgene of non-fluorescent

Fig. 1. Development of CRISPR-mediated gene targeting in Tribolium. (A) The Pig-19 transgenic line, used to test gene targeting, carries an insertion of3xP3-EGFP which is activated by an endogenous muscle enhancer. The eGFP1 gRNA targets the EGFP coding sequence (red arrowhead). (B) Pig-19 larvashowing strong EGFP fluorescence in muscles. (C) Pig-19G0 larvae, arising from embryos injected with eGFP1 gRNA andCas9mRNA, showing mosaic loss ofEGFP fluorescence. (D) Sequencing of the EGFP transgene from G1 larvae that lack fluorescence reveals small deletions at the eGFP1 target site (in gray). (E)Maps of the bhsp68-Cas9, U6a-BsaI-gRNA and U6b-BsaI-gRNA constructs, used to drive expression of gRNAs and Cas9, respectively. Detailed views of theU6a-BsaI-gRNA and U6b-BsaI-gRNA cloning sites are shown, indicating the sites where new targeting sequences can be inserted (green lines).

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larvae confirmed the presence of indels at the eGFP1 target site(Fig. 1D).In our hands, CRISPR/Cas9 performed more efficiently than a

pair of zinc-finger nucleases that are known to target EGFP(Porteus, 2006): no somatic loss of EGFP was detected upon ZFNmRNA injection in Pig-19 embryos and 26% of these G0 animals(5 out of 19) produced non-fluorescent G1 progeny.

Endogenous promoters to drive guide RNA and Cas9expressionUsing promoters to express gRNA and Cas9 from plasmids ortransgenes can be a useful alternative to delivering in vitrotranscribed gRNA and Cas9, in terms of ease of use, efficiencyand robustness. The bacteriophage promoters used to in vitrotranscribe gRNAs also impose some additional constraints on thetarget sequence (reviewed by Gilles and Averof, 2014). Wetherefore set out to identify appropriate Tribolium promoters.First, we tested U6 promoters as a means of expressing gRNAs.

U6 snRNAs are transcribed by RNA polymerase III, which issuitable for expressing small RNAs with precisely defined 5′ and 3′ends, such as gRNAs (Cong et al., 2013; Dickinson et al., 2013;Jinek et al., 2013;Mali et al., 2013b; Ren et al., 2013). U6 promotersgenerate transcripts that start with a G and can thus be used tooptimally target sequences that conform to G-N16-19-NGG (inpractice, a mismatched G at the 5′ end of the gRNA can be tolerated;Fu et al., 2014; Hwang et al., 2013a).We identified three U6 snRNA genes in the Tribolium genome.

We cloned two putative promoter fragments, named U6a and U6b,

spanning ∼400 bp upstream of the transcription start site of two ofthose genes (seeMaterials andMethods).We placed these fragmentsupstream of the eGFP1 gRNA, generating plasmids p(U6a-eGFP1.gRNA) and p(U6b-eGFP1.gRNA), and tested their activity byinjecting them into Pig-19 embryos together with Cas9 mRNA.

Overall, we found that the U6-gRNA plasmids gave similartargeting efficiencies and improved survival compared with in vitrosynthesized gRNA (Table 1, rows 3, 4). Somatic loss of EGFP inmuscles was observed in 78% of G0 larvae. Knockout of EGFP wasobserved in the progeny of 91-100% of G0 animals with U6-drivengRNA versus 71% with in vitro gRNA (Table 1, rows 2-4). Thus,the Tribolium U6a andU6b promoters are capable of driving gRNAexpression and mediating efficient gene targeting.

To facilitate the expression of different guide RNAs under theseU6 promoters we designed plasmids p(U6a-BsaI-gRNA) andp(U6b-BsaI-gRNA), containing two BsaI restriction sites located5′ of the gRNA scaffold. Complementary oligonucleotides carryingappropriate overhangs can be used to introduce the chosen targetingsequence in a single cloning step (see Fig. 1E; www.averof-lab.org/tools/Tribolium_CRISPR.php).

Next, we generated a Cas9 helper plasmid, p(bhsp68-Cas9),bearing the Streptococcus pyogenes Cas9 coding sequencedownstream of the Tribolium hsp68 core promoter (Schinko et al.,2012), which drives low levels of expression constitutively.We testedthe activity of this plasmid by co-injecting it with p(U6b-eGFP1.gRNA) in Pig-19 embryos. Sixty-two percent of G0 larvae showedsomatic loss of EGFP in muscles, and 83% of outcrossed G0 animalsproduced non-fluorescent progeny (Table 1, row 5).

Table 1. Results of CRISPR experiments

G0 G1

gRNA source(ng/µl)

Cas9source(ng/µl)

Knock-intemplate(ng/µl)

Number ofembryosinjected

Number ofsurvivinglarvae (%)

Number ofmosaicknockoutlarvae (%)

Number ofmosaicknock-inlarvae (%)

Number ofG0 animalscrossed

Number ofknockoutfounders(%)

Number ofknock-infounders(%)

eGFP1 gRNA(30)

Cas9mRNA(390)

– 562* 54 (10)* 43 (80)* – – – –

eGFP1 gRNA(30)

Cas9mRNA(390)

– 441 55 (12) 39 (71) – 38 27 (71) –

p(U6a-eGFP1.gRNA) (500)

Cas9mRNA(180)

– 190 111 (58) 87 (78) – 11 10 (91) –

p(U6b-eGFP1.gRNA) (500)

Cas9mRNA(180)

– 295 123 (42) 97 (79) – 17 17 (100) –

p(U6b-eGFP1.gRNA) (500)

p(bhsp68-Cas9)(1000)

– 166 34 (20) 21 (62) – 18 15 (83) –

p(U6b-eGFP1.gRNA) (500)

p(bhsp68-Cas9)(500)

pBac(3xP3-DsRedfa)(500)

1866‡ 444 (24)‡ 246 (55)‡ 61 (14)‡ 315‡ 230 (73)‡ 18 (6)‡

p(U6b-eGFP1.gRNA) (500)

– pBac(3xP3-DsRedfa)(500)

1114§ 242 (22)§ 0 (0)§ 0 (0)§ – – –

p(U6b-eGFP1.gRNA)(500)¶

p(bhsp68-Cas9)(1000)¶

pBac(3xP3-DsRedfa)(500)¶

191 23 (12) 15 (65) 4 (17) 14 13 (93) 1 (7)

*Data from two independent experiments.‡Data from five independent experiments.§Data from four independent experiments.¶Co-injected with 200 ng/μl dsRNA for DNA ligase 4.

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These results establish plasmid injection as an efficient meansfor the routine delivery of Cas9 and gRNAs in Tribolium. Therelevant plasmids are available from www.addgene.com (EMBL/Genbank sequence accession numbers KR732918, KR732919 andKR732920).

Targeting of endogenous lociTo demonstrate targeting of endogenous loci in the Triboliumgenome, we generated a gRNA targeting the coding sequence ofTribolium E-cadherin (gene TC013570) in plasmid p(U6b-Ecadherin.gRNA), as described above. We co-injected thisplasmid with p(bhsp68-Cas9) in embryos of the vermilionwhite

strain. Previous work had shown that embryonic RNAi for E-cadherin produces embryos that fail to complete dorsal closure,which can be observed in larval cuticle preps (A.F.G. andG. Bucher, unpublished). In our experiment, 53% (46 out of 86)of injected embryos developed a cuticle and 24% of those larvae (11out of 46) showed dorsal closure defects (Fig. 2A,B). PCRamplification of the target locus from two G0 larvae revealedCRISPR-induced deletions at the target locus (Fig. 2C).We performed similar experiments targeting the coding sequence

of Tribolium twist and found that 99% of the G0 embryos (244 outof 247) failed to hatch. PCR amplification of the target locus fromtwo G0 animals revealed several different mutations surroundingthe target site (Fig. 2D).

CRISPR-induced homology directed knock-insTaking advantage of the Pig-19 line and the eGFP1 gRNA, wetested the efficiency of CRISPR in generating knock-ins inTribolium. Specifically, we attempted to replace the 3xP3-EGFPtransgene in the Pig-19 line by a 3xP3-DsRed transgene, exploiting

the arms of the piggyBac transposon flanking the transgene to guideHDR; the homologous arms are 0.7 and 1 kb in length (Fig. 3A).

We injected p(U6b-eGFP1.gRNA) and p(bhsp68-Cas9) togetherwith pBac(3xP3-DsRedaf ) (Horn et al., 2002) as a template forHDR, and assessed EGFP and DsRed fluorescence in G0 and G1larvae. To assess reproducibility, we repeated this experiment fivetimes (the pooled results are shown in Table 1, row 6).

Two weeks after injection, somatic loss of EGFP was seen in46-70% of G0 larvae and DsRed fluorescence in muscle fiberswas observed in 8-20% of G0 larvae (Fig. 3B). Crossing thesurviving G0 animals to vermilionwhite beetles, we found that 24-77% of crosses produced G0 larvae lacking EGFP expression and0-10% of crosses produced larvae in which EGFP fluorescencewas replaced by DsRed fluorescence in the muscles and in the3xP3-driven pattern. DsRed-expressing G1 larvae were obtainedin four out of five experiments. The proportion of DsRed-expressing larvae emerging from each cross ranged from 2% to34%, among ∼50 screened larvae. Sequencing the entire Pig-19insertion locus from a G1 larva confirmed the seamlesshomology-driven replacement of the 3xP3-EGFP transgene by3xP3-DsRed. No somatic targeting was seen in the absence ofCas9 (Table 1, row 7).

As noted for knockouts, somatic targeting in the G0 generationcorrelated with the likelihood of targeting in G1: across all fiveexperiments, the fraction of G0 animals giving rise to DsRed-expressing G1 larvae was 12% among G0 animals with mosaicexpression of DsRed (6 out of 50) versus 5% among non-DsRed-expressing G0 animals (12 out of 265).

Experiments inDrosophila show that mutations in DNA ligase 4,an enzyme required for NHEJ, can lead to an increase in theefficiency of HDR-mediated knock-ins (Beumer et al., 2008, 2013).

Fig. 2. Targeting of TriboliumE-cadherin and twist. (A,B) Cuticlepreparations of a wild-type larva (A) and aG0 larva arising fromCRISPR targeting ofTribolium E-cadherin (B). The dorsalclosure defect (asterisk in B) iscomparable to the phenotype seen afterE-cadherin RNAi. (C,D) Sequences ofE-cadherin and twist from G0 larvaetargeted with the corresponding gRNAs,revealing mutations at the target site(in gray).

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To test whether RNAi knockdown of DNA ligase 4 can improveknock-in efficiency in Tribolium, we performed the DsRed knock-in experiment with the addition of double-stranded RNA forTribolium DNA ligase 4 in the injection mix. Knock-in efficiencywas not significantly improved: 17% of G0 larvae showed mosaicexpression of DsRed and 7% of crossed G0 animals producedDsRed-expressing G1 larvae (Table 1, row 8). Maternally depositedDNA ligase 4 protein, which is unaffected by embryonic RNA, mayaccount for this result.

Excision and replacement of genomic regionsUsing the same tools, we investigated the feasibility of deleting aspecific genomic fragment by using pairs of double-strand breaks,and of replacing that fragment by an exogenous DNA construct.This approach could have numerous applications, such as deletingsingle exons or re-purposing transgenes (such as gene traps) at theiroriginal site of insertion.To test the approach, we designed gRNAs targeting the left and

the right arms of the piggyBac transposon ( pBacL and pBacR). Inthe genome of Pig-19 beetles these sites delimit a fragment of∼2 kbthat includes the 3xP3-EGFP transgene. The replacement transgene,3xP3-DsRed, is flanked by the same target sites in plasmid pBac(3xP3-DsRedaf ) (Horn et al., 2002) and is therefore targeted by thesame gRNAs (Fig. 4A). Plasmids expressing gRNAs for pBacL andpBacRwere injected together with p(bhsp68-Cas9) and pBac(3xP3-DsRedaf ). Forty-seven percent of the surviving G0 animals (22 outof 47) displayed mosaic DsRed fluorescence in muscles, sometimesaccompanied by loss of EGFP, indicating integration of DsRed inthe Pig-19 locus.Thirty-four percent of G0 animals outcrossed to vermilionwhite

beetles produced some progeny that had lost EGFP fluorescence. Of

those, 22% G0 animals had some progeny in which the loss ofEGFP was accompanied by DsRed fluorescence in muscles,indicating the replacement of the EGFP transgene by DsRed, and12% G0 animals had some progeny that had both DsRed and EGFPfluorescence in muscles, presumably resulting from the insertion ofthe 3xP3-DsRed transgene at the pBacL or the pBacR target sitewithout concomitant deletion of 3xP3-EGFP (Table 2).

To investigate the nature of these targeting events, we amplifiedand sequenced the genomic DNA of selected G1 animals at the Pig-19 insertion locus. Among the sequenced G1 animals that had lostEGFP fluorescence, six out of seven carried an excision of the 2-kbfragment delimited by the two gRNA target sites (Fig. 4B; theseventh carried a partial inversion and deletion of the transgene). Allthe G1 animals with DsRed fluorescence accompanied by loss ofEGFP (4 out of 4) had replaced the 3xP3-EGFP transgene by 3xP3-DsRed, in the expected orientation (Fig. 4C). The integration eventsseamlessly restored the pBacL and pBacR sequence in six out of theeight insertion breakpoints, suggesting a precise repair by HDR; theother two breakpoints carried 3-bp deletions, which suggests thatthey were repaired by NHEJ. Some G1 larvae (5 out of 18) failed toproduce the expected PCR bands, possibly reflecting more complexdeletion and rearrangement events.

ConclusionsOur experiments demonstrate that CRISPR technology is aneffective method for generating targeted knockouts, knock-insand deletions in Tribolium. Deletion of genomic fragments drivenby pairs of double-strand breaks, as demonstrated here, can be usedto test the function of specific exons and putative cis-regulatoryelements. Transgene replacement, shown for Pig-19, can be used toexploit the large collection of transgene insertions that already exist

Fig. 3. CRISPR-mediated gene knock-ins. (A) Illustration of a CRISPR-induced knock-in at the Pig-19 locus. The double-strand break introduced by eGFP1gRNA and Cas9 is repaired from a plasmid template with 0.7- and 1-kb homology arms flanking a 3xP3-DsRed transgene. Homology-directed repair leads to thereplacement of 3xP3-EGFP by 3xP3-DsRed in the target locus. (B) Pig19 G0 larva with clones of DsRed-expressing muscle.

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in Tribolium (Lorenzen et al., 2003; Trauner et al., 2009) to producea wider range of genetic tools, such as GAL4 drivers (Schinko et al.,2010).In this study, we did not investigate potential off-target effects of

CRISPR. As in other experimental systems, we expect thatunintended targets will occasionally be hit in the Triboliumgenome (Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013;Pattanayak et al., 2013). Equally, we anticipate that thetechnical solutions developed to reduce and control for theseeffects (discussed by Gilles and Averof, 2014) will also apply toTribolium.

The promoters and constructs that we established for the expressionof Cas9 and gRNAs facilitate the implementation of CRISPR in thisspecies.We hope that these tools will also help to establish transgene-based applications of CRISPR, such as inducible and tissue-specifictargeting (Port et al., 2014), and effectors for manipulating thefunction of genes in their native context (Mali et al., 2013a).

MATERIALS AND METHODSCas9 mRNA and gRNA synthesisCas9 mRNA was in vitro transcribed from Addgene plasmid MLM3613(Addgene; Hwang et al., 2013b) linearized with PmeI, using the

Fig. 4. Targeted excision and replacement of genomic fragments. (A) Illustration of the transgene excision and replacement, using pairs of gRNAs targetingthe ends of the fragment to be excised and of the new fragment to be inserted. (B,C) Sequencing of the Pig-19 locus from individual G1 larvae: from six larvaewith no fluorescence, demonstrating excision of the EGFP transgene (B), and from four larvae with DsRed fluorescence, demonstrating replacement of3xP3-EGFP by 3xP3-DsRed (C).

Table 2. Results of CRISPR-mediated excision and replacement experiments

gRNA and Cas9 injected (ng/µl)Replacementtemplate (ng/µl)

Number ofembryos injected

Number of G0animals crossed

Number of G0founders (%)

Events scoredamong G1 animals

p(U6b-pBacL.gRNA) and p(U6b-pBacR.gRNA) (200 each), p(bhsp68-Cas9)(500)

pBac(3xP3-DsRedaf) (250)

170 41 27 (66) No targeting14 (34)* EGFP excision9 (22)* EGFP-to-DsRed

replacement5 (12)* DsRed insertion

(with EGFP)

*Individual G0 founders produced different types of targeting events in G1 animals, so these categories overlap.

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mMESSAGEmMACHINE T7 Ultra kit (Ambion). The eGFP1 guide RNAwas in vitro transcribed from a plasmid (Auer et al., 2014) linearized withDraI, using the T7 Megascript kit (Ambion).

Cas9 helper plasmidThe Cas9 coding sequence was amplified from plasmid MLM3613(Addgene; Hwang et al., 2013b) using primers AG045 and AG046,which carry AgeI and NotI restriction sites (supplementary materialTables S1 and S2). The PCR product was cloned into the AgeI and NotIsites of pSLfa(bhsp68-NLS.EGFP.hsp3′UTR)fa (Schinko et al., 2012),replacing EGFP by the Cas9 coding sequence.

U6 promoter and gRNA plasmidsThree U6 snRNA genes were identified in the Tribolium genome bysequence similarity with the Drosophila U6 snRNA, using BLAST. Weamplified ∼400 bp of upstream sequences from two of these genes,designated U6a and U6b. Using overlapping PCR primers (supplementarymaterial Table S1), we cloned each of these sequences upstream of theeGFP1 guide RNA (Auer et al., 2014) or of a gRNA scaffold with BsaIcloning sites for inserting new targeting sequences (Fig. 1E; Hwang et al.,2013b), in pBluescript-II-KS+ and pBME-Amp (BioMatik) plasmids,respectively (supplementary material Table S2). A U6 transcriptionterminator (T6) was included at the 3′end of the gRNA scaffold.

CRISPR target sites in the Tribolium twist and the E-Cadherin codingsequences were identified using the flyCRISPR target finder (http://tools.flycrispr.molbio.wisc.edu/targetFinder/; Gratz et al., 2014). Pairs ofcomplementary oligonucleotides were designed for each target site,leaving appropriate overhangs for inserting these sequences in the BsaIsite of p(U6b-BsaI-gRNA) (oligo design tool available at www.averof-lab.org/tools/Tribolium_CRISPR.php). The oligonucleotides were annealedand cloned into p(U6b-BsaI-gRNA) digested with BsaI.

Tribolium DNA ligase 4The Tribolium DNA ligase 4 ortholog (gene TC012219) was identified bysequence similarity with Drosophila DNA ligase 4 protein, using BLAST.A 2-kb fragment containing the largest exon was cloned from genomicDNA, using primers AG001 and AG002 (supplementary materialTable S1), into pGEM-T-Easy (Promega). Double-stranded RNA wasproduced as described previously (Posnien et al., 2009).

Tribolium injection and molecular analysisTribolium embryos were injected at the syncytial blastoderm stage andhandled as described previously (Berghammer et al., 2009). The injectedconcentrations of Cas9, gRNA and knock-in templates are indicated inTable 1. Plasmids were prepared using the NucleoBond Xtra Midi kit(Macherey-Nagel) and injected in supercoiled form.

The loci targeted by CRISPR were amplified by PCR (primers listed insupplementary material Table S1), cloned and sequenced.

AcknowledgementsWe thank Martin Klingler for discussions on the application of CRISPR in Tribolium;Max Telford, Meriem Takarli and Josh Coulcher for preliminary experiments usingzinc-finger nucleases; Thomas Auer, Filippo del Bene and Marco Grillo fordiscussing targeting strategies; and Gregor Bucher for sharing Tribolium stocks.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsA.F.G. and J.B.S. performed the experiments and analyzed the results; A.F.G.,J.B.S. and M.A. planned the study and wrote the paper.

FundingThis work was supported by a PhD fellowship from the Universite Claude BernardLyon 1 (A.F.G.); a travel grant from the Boehringer Ingelheim Foundation (A.F.G.); apost-doctoral fellowship of the Fondation pour la Recherche Medicale (J.B.S.); andby a grant from the Agence Nationale de la Recherche [ANR-12-CHEX-0001-01].

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.125054/-/DC1

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