© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2832-2839 doi:10.1242/dev.125054
RESEARCH REPORT
TECHNIQUES AND RESOURCES
Efficient CRISPR-mediated gene targeting and transgene
replacement in the beetle Tribolium castaneum
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
1
Institut de Gé nomique Fonctionnelle de Lyon (IGFL), École Normale Supé rieure de
2
Lyon, 46 Allé e d’Italie, Lyon 69264, France. École doctorale BMIC, Université
3
Claude Bernard, Lyon 1, France. Centre National de la Recherche Scientifique
(CNRS), France.
*Authors for correspondence ([email protected]; [email protected])
Received 7 April 2015; Accepted 29 June 2015
2832
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 cells’ endogenous 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 homologydirected repair (HDR) provides an opportunity to introduce
specific changes into the locus via an engineered template. Zincfinger 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 5′ end 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 (N17-20)
at the 5′ end of a gRNA, it is possible to generate nucleases targeting
any sequence that conforms to N17-20NGG (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 organism’s 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
DEVELOPMENT
Anna F. Gilles1,2, *, Johannes B. Schinko1 and Michalis Averof1,3,*
screens conducted in the 1980s (Beeman et al., 1989; Sulston and
Anderson, 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), RNAimediated knockdown (Bucher et al., 2002; Posnien et al., 2009),
enhancer trapping (Lorenzen et al., 2003; Trauner et al., 2009), heatshock 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 is
sequenced and well annotated (Kim et al., 2010; Richards et al.,
2008). Based on these tools and resources, Tribolium castaneum has
become the most advanced genetically tractable insect model after
Drosophila.
Establishing efficient gene targeting in Tribolium brings to this
model a more precise tool for reverse genetics, a directed approach
for generating new markers and GAL4 drivers, and the opportunity
to generate balancer chromosomes (for a more comprehensive
discussion on the potential uses of CRISPR in emerging models, see
Gilles and Averof, 2014).
RESULTS AND DISCUSSION
Targeting of a genomic EGFP insertion using CRISPR
We established an efficient assay for scoring gene targeting events
in Tribolium, making use of the enhancer trap line Pig-19 (Lorenzen
et al., 2003). Pig-19 is homozygous for a single insertion of
pBac(3xP3-EGFP) in the Tribolium genome. Transgenic animals
have strong enhanced green fluorescent protein (EGFP)
Development (2015) 142, 2832-2839 doi:10.1242/dev.125054
fluorescence in larval, pupal and adult muscles (Fig. 1A,B). As a
proof of concept for the functionality of CRISPR/Cas9 in
Tribolium, we injected Pig-19 embryos with in vitro transcribed
eGFP1 gRNA, targeting the EGFP coding sequence 116 bp after
the start codon (Auer et al., 2014), together with capped Cas9
mRNA.
Three days after injection, the freshly hatched larvae were
scrutinized for loss of EGFP expression in muscles. EGFP was
visibly altered in 70-80% of the larvae (Table 1, rows 1, 2). The
mildest phenotypes were small patches of non-fluorescent muscles,
the strongest were loss of EGFP fluorescence in muscles of all
segments (Fig. 1C). The loss of EGFP from muscles indicates biallelic targeting of EGFP in all the nuclei of the multinucleated
muscle fiber, a sign of highly efficient somatic gene knockout.
To assess the frequency of germline targeting events, we raised
injected (G0) animals and crossed them out to adult vermilionwhite
beetles (Lorenzen et al., 2002). We then screened the progeny of
each cross (G1) and determined the number of G0 animals that gave
rise to non-fluorescent progeny (founders). Seventy-one percent of
fertile crosses produced non-fluorescent progeny (Table 1, row 2).
G0 animals showing mosaic loss of EGFP were more likely to
produce non-fluorescent offspring (21 out of 22) compared with G0
animals with normal EGFP fluorescence (6 out of 16), suggesting
that somatic targeting in G0 larvae may be a useful indicator of
germline targeting in these animals.
The proportion of non-fluorescent larvae emerging from
each cross was variable, ranging from 8% to 100% of G1
animals. 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 of
3xP3-EGFP which is activated by an endogenous muscle enhancer. The eGFP1 gRNA targets the EGFP coding sequence (red arrowhead). (B) Pig-19 larva
showing strong EGFP fluorescence in muscles. (C) Pig-19 G0 larvae, arising from embryos injected with eGFP1 gRNA and Cas9 mRNA, showing mosaic loss of
EGFP 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 the
U6a-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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DEVELOPMENT
RESEARCH REPORT
RESEARCH REPORT
Development (2015) 142, 2832-2839 doi:10.1242/dev.125054
Table 1. Results of CRISPR experiments
G0
gRNA source
(ng/µl)
eGFP1 gRNA
(30)
eGFP1 gRNA
(30)
p(U6a-eGFP1.
gRNA) (500)
p(U6b-eGFP1.
gRNA) (500)
p(U6b-eGFP1.
gRNA) (500)
p(U6b-eGFP1.
gRNA) (500)
p(U6b-eGFP1.
gRNA) (500)
p(U6b-eGFP1.
gRNA)
(500)¶
G1
Cas9
source
(ng/µl)
Knock-in
template
(ng/µl)
Number of
embryos
injected
Number of
surviving
larvae (%)
Number of
mosaic
knockout
larvae (%)
Number of
mosaic
knock-in
larvae (%)
Number of
G0 animals
crossed
Number of
knockout
founders
(%)
Number of
knock-in
founders
(%)
Cas9
mRNA
(390)
Cas9
mRNA
(390)
–
562*
54 (10)*
43 (80)*
–
–
–
–
–
441
55 (12)
39 (71)
–
38
27 (71)
–
Cas9
mRNA
(180)
Cas9
mRNA
(180)
p(bhsp68Cas9)
(1000)
–
190
111 (58)
87 (78)
–
11
10 (91)
–
–
295
123 (42)
97 (79)
–
17
17 (100)
–
–
166
34 (20)
21 (62)
–
18
15 (83)
–
p(bhsp68Cas9)
(500)
–
pBac(3xP3DsRedfa)
(500)
pBac(3xP3DsRedfa)
(500)
1866‡
444 (24)‡
246 (55)‡
61 (14)‡
315‡
230 (73)‡
18 (6)‡
1114§
242 (22)§
0 (0)§
0 (0)§
–
–
–
p(bhsp68Cas9)
(1000)¶
pBac(3xP3DsRedfa)
(500)¶
191
23 (12)
15 (65)
4 (17)
14
13 (93)
1 (7)
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 ZFN
mRNA 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 Cas9
expression
Using promoters to express gRNA and Cas9 from plasmids or
transgenes can be a useful alternative to delivering in vitro
transcribed gRNA and Cas9, in terms of ease of use, efficiency
and robustness. The bacteriophage promoters used to in vitro
transcribe gRNAs also impose some additional constraints on the
target sequence (reviewed by Gilles and Averof, 2014). We
therefore 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 is
suitable 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 promoters
generate transcripts that start with a G and can thus be used to
optimally target sequences that conform to G-N16-19-NGG (in
practice, 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,
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spanning ∼400 bp upstream of the transcription start site of two of
those genes (see Materials and Methods). We placed these fragments
upstream of the eGFP1 gRNA, generating plasmids p(U6a-eGFP1.
gRNA) and p(U6b-eGFP1.gRNA), and tested their activity by
injecting them into Pig-19 embryos together with Cas9 mRNA.
Overall, we found that the U6-gRNA plasmids gave similar
targeting efficiencies and improved survival compared with in vitro
synthesized gRNA (Table 1, rows 3, 4). Somatic loss of EGFP in
muscles was observed in 78% of G0 larvae. Knockout of EGFP was
observed in the progeny of 91-100% of G0 animals with U6-driven
gRNA versus 71% with in vitro gRNA (Table 1, rows 2-4). Thus,
the Tribolium U6a and U6b promoters are capable of driving gRNA
expression and mediating efficient gene targeting.
To facilitate the expression of different guide RNAs under these
U6 promoters we designed plasmids p(U6a-BsaI-gRNA) and
p(U6b-BsaI-gRNA), containing two BsaI restriction sites located
5′ of the gRNA scaffold. Complementary oligonucleotides carrying
appropriate overhangs can be used to introduce the chosen targeting
sequence 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 sequence
downstream of the Tribolium hsp68 core promoter (Schinko et al.,
2012), which drives low levels of expression constitutively. We tested
the activity of this plasmid by co-injecting it with p(U6b-eGFP1.
gRNA) in Pig-19 embryos. Sixty-two percent of G0 larvae showed
somatic loss of EGFP in muscles, and 83% of outcrossed G0 animals
produced non-fluorescent progeny (Table 1, row 5).
DEVELOPMENT
*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.
RESEARCH REPORT
Development (2015) 142, 2832-2839 doi:10.1242/dev.125054
These results establish plasmid injection as an efficient means
for the routine delivery of Cas9 and gRNAs in Tribolium. The
relevant plasmids are available from www.addgene.com (EMBL/
Genbank sequence accession numbers KR732918, KR732919 and
KR732920).
Targeting of endogenous loci
To demonstrate targeting of endogenous loci in the Tribolium
genome, we generated a gRNA targeting the coding sequence of
Tribolium E-cadherin (gene TC013570) in plasmid p(U6bEcadherin.gRNA), as described above. We co-injected this
plasmid with p(bhsp68-Cas9) in embryos of the vermilionwhite
strain. Previous work had shown that embryonic RNAi for Ecadherin produces embryos that fail to complete dorsal closure,
which can be observed in larval cuticle preps (A.F.G. and
G. Bucher, unpublished). In our experiment, 53% (46 out of 86)
of injected embryos developed a cuticle and 24% of those larvae (11
out of 46) showed dorsal closure defects (Fig. 2A,B). PCR
amplification of the target locus from two G0 larvae revealed
CRISPR-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 out
of 247) failed to hatch. PCR amplification of the target locus from
two G0 animals revealed several different mutations surrounding
the target site (Fig. 2D).
CRISPR-induced homology directed knock-ins
Taking advantage of the Pig-19 line and the eGFP1 gRNA, we
tested the efficiency of CRISPR in generating knock-ins in
Tribolium. Specifically, we attempted to replace the 3xP3-EGFP
transgene in the Pig-19 line by a 3xP3-DsRed transgene, exploiting
the arms of the piggyBac transposon flanking the transgene to guide
HDR; the homologous arms are 0.7 and 1 kb in length (Fig. 3A).
We injected p(U6b-eGFP1.gRNA) and p(bhsp68-Cas9) together
with pBac(3xP3-DsRedaf ) (Horn et al., 2002) as a template for
HDR, and assessed EGFP and DsRed fluorescence in G0 and G1
larvae. To assess reproducibility, we repeated this experiment five
times (the pooled results are shown in Table 1, row 6).
Two weeks after injection, somatic loss of EGFP was seen in
46-70% of G0 larvae and DsRed fluorescence in muscle fibers
was observed in 8-20% of G0 larvae (Fig. 3B). Crossing the
surviving G0 animals to vermilionwhite beetles, we found that 2477% of crosses produced G0 larvae lacking EGFP expression and
0-10% of crosses produced larvae in which EGFP fluorescence
was replaced by DsRed fluorescence in the muscles and in the
3xP3-driven pattern. DsRed-expressing G1 larvae were obtained
in four out of five experiments. The proportion of DsRedexpressing larvae emerging from each cross ranged from 2% to
34%, among ∼50 screened larvae. Sequencing the entire Pig-19
insertion locus from a G1 larva confirmed the seamless
homology-driven replacement of the 3xP3-EGFP transgene by
3xP3-DsRed. No somatic targeting was seen in the absence of
Cas9 (Table 1, row 7).
As noted for knockouts, somatic targeting in the G0 generation
correlated with the likelihood of targeting in G1: across all five
experiments, the fraction of G0 animals giving rise to DsRedexpressing G1 larvae was 12% among G0 animals with mosaic
expression of DsRed (6 out of 50) versus 5% among non-DsRedexpressing G0 animals (12 out of 265).
Experiments in Drosophila show that mutations in DNA ligase 4,
an enzyme required for NHEJ, can lead to an increase in the
efficiency of HDR-mediated knock-ins (Beumer et al., 2008, 2013).
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DEVELOPMENT
Fig. 2. Targeting of Tribolium
E-cadherin and twist. (A,B) Cuticle
preparations of a wild-type larva (A) and a
G0 larva arising from CRISPR targeting of
Tribolium E-cadherin (B). The dorsal
closure defect (asterisk in B) is
comparable to the phenotype seen after
E-cadherin RNAi. (C,D) Sequences of
E-cadherin and twist from G0 larvae
targeted with the corresponding gRNAs,
revealing mutations at the target site
(in gray).
RESEARCH REPORT
Development (2015) 142, 2832-2839 doi:10.1242/dev.125054
To test whether RNAi knockdown of DNA ligase 4 can improve
knock-in efficiency in Tribolium, we performed the DsRed knockin experiment with the addition of double-stranded RNA for
Tribolium DNA ligase 4 in the injection mix. Knock-in efficiency
was not significantly improved: 17% of G0 larvae showed mosaic
expression of DsRed and 7% of crossed G0 animals produced
DsRed-expressing G1 larvae (Table 1, row 8). Maternally deposited
DNA ligase 4 protein, which is unaffected by embryonic RNA, may
account for this result.
Excision and replacement of genomic regions
Using the same tools, we investigated the feasibility of deleting a
specific 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 deleting
single exons or re-purposing transgenes (such as gene traps) at their
original site of insertion.
To test the approach, we designed gRNAs targeting the left and
the right arms of the piggyBac transposon ( pBacL and pBacR). In
the genome of Pig-19 beetles these sites delimit a fragment of ∼2 kb
that 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 the
same gRNAs (Fig. 4A). Plasmids expressing gRNAs for pBacL and
pBacR were injected together with p(bhsp68-Cas9) and pBac(3xP3DsRedaf ). Forty-seven percent of the surviving G0 animals (22 out
of 47) displayed mosaic DsRed fluorescence in muscles, sometimes
accompanied by loss of EGFP, indicating integration of DsRed in
the Pig-19 locus.
Thirty-four percent of G0 animals outcrossed to vermilionwhite
beetles produced some progeny that had lost EGFP fluorescence. Of
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those, 22% G0 animals had some progeny in which the loss of
EGFP was accompanied by DsRed fluorescence in muscles,
indicating the replacement of the EGFP transgene by DsRed, and
12% G0 animals had some progeny that had both DsRed and EGFP
fluorescence in muscles, presumably resulting from the insertion of
the 3xP3-DsRed transgene at the pBacL or the pBacR target site
without concomitant deletion of 3xP3-EGFP (Table 2).
To investigate the nature of these targeting events, we amplified
and sequenced the genomic DNA of selected G1 animals at the Pig19 insertion locus. Among the sequenced G1 animals that had lost
EGFP fluorescence, six out of seven carried an excision of the 2-kb
fragment delimited by the two gRNA target sites (Fig. 4B; the
seventh carried a partial inversion and deletion of the transgene). All
the G1 animals with DsRed fluorescence accompanied by loss of
EGFP (4 out of 4) had replaced the 3xP3-EGFP transgene by 3xP3DsRed, in the expected orientation (Fig. 4C). The integration events
seamlessly restored the pBacL and pBacR sequence in six out of the
eight insertion breakpoints, suggesting a precise repair by HDR; the
other two breakpoints carried 3-bp deletions, which suggests that
they were repaired by NHEJ. Some G1 larvae (5 out of 18) failed to
produce the expected PCR bands, possibly reflecting more complex
deletion and rearrangement events.
Conclusions
Our experiments demonstrate that CRISPR technology is an
effective method for generating targeted knockouts, knock-ins
and deletions in Tribolium. Deletion of genomic fragments driven
by pairs of double-strand breaks, as demonstrated here, can be used
to test the function of specific exons and putative cis-regulatory
elements. Transgene replacement, shown for Pig-19, can be used to
exploit the large collection of transgene insertions that already exist
DEVELOPMENT
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 eGFP1
gRNA 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 the
replacement of 3xP3-EGFP by 3xP3-DsRed in the target locus. (B) Pig19 G0 larva with clones of DsRed-expressing muscle.
RESEARCH REPORT
Development (2015) 142, 2832-2839 doi:10.1242/dev.125054
Fig. 4. Targeted excision and replacement of genomic fragments. (A) Illustration of the transgene excision and replacement, using pairs of gRNAs targeting
the 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 larvae
with no fluorescence, demonstrating excision of the EGFP transgene (B), and from four larvae with DsRed fluorescence, demonstrating replacement of
3xP3-EGFP by 3xP3-DsRed (C).
The promoters and constructs that we established for the expression
of Cas9 and gRNAs facilitate the implementation of CRISPR in this
species. We hope that these tools will also help to establish transgenebased applications of CRISPR, such as inducible and tissue-specific
targeting (Port et al., 2014), and effectors for manipulating the
function of genes in their native context (Mali et al., 2013a).
MATERIALS AND METHODS
Cas9 mRNA and gRNA synthesis
Cas9 mRNA was in vitro transcribed from Addgene plasmid MLM3613
(Addgene; Hwang et al., 2013b) linearized with PmeI, using the
Table 2. Results of CRISPR-mediated excision and replacement experiments
gRNA and Cas9 injected (ng/µl)
p(U6b-pBacL.gRNA) and p(U6b-pBacR.
gRNA) (200 each), p(bhsp68-Cas9)
(500)
Replacement
template (ng/µl)
Number of
embryos injected
Number of G0
animals crossed
Number of G0
founders (%)
Events scored
among G1 animals
pBac(3xP3DsRedaf) (250)
170
41
27 (66)
14 (34)*
9 (22)*
No targeting
EGFP excision
EGFP-to-DsRed
replacement
DsRed insertion
(with EGFP)
5 (12)*
*Individual G0 founders produced different types of targeting events in G1 animals, so these categories overlap.
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DEVELOPMENT
in Tribolium (Lorenzen et al., 2003; Trauner et al., 2009) to produce
a 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 that
unintended targets will occasionally be hit in the Tribolium
genome (Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013;
Pattanayak et al., 2013). Equally, we anticipate that the
technical solutions developed to reduce and control for these
effects (discussed by Gilles and Averof, 2014) will also apply to
Tribolium.
RESEARCH REPORT
Cas9 helper plasmid
The 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 material
Tables S1 and S2). The PCR product was cloned into the AgeI and NotI
sites of pSLfa(bhsp68-NLS.EGFP.hsp3′UTR)fa (Schinko et al., 2012),
replacing EGFP by the Cas9 coding sequence.
U6 promoter and gRNA plasmids
Three U6 snRNA genes were identified in the Tribolium genome by
sequence similarity with the Drosophila U6 snRNA, using BLAST. We
amplified ∼400 bp of upstream sequences from two of these genes,
designated U6a and U6b. Using overlapping PCR primers (supplementary
material Table S1), we cloned each of these sequences upstream of the
eGFP1 guide RNA (Auer et al., 2014) or of a gRNA scaffold with BsaI
cloning 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 transcription
terminator (T6) was included at the 3′end of the gRNA scaffold.
CRISPR target sites in the Tribolium twist and the E-Cadherin coding
sequences were identified using the flyCRISPR target finder (http://tools.
flycrispr.molbio.wisc.edu/targetFinder/; Gratz et al., 2014). Pairs of
complementary oligonucleotides were designed for each target site,
leaving appropriate overhangs for inserting these sequences in the BsaI
site of p(U6b-BsaI-gRNA) (oligo design tool available at www.averof-lab.
org/tools/Tribolium_CRISPR.php). The oligonucleotides were annealed
and cloned into p(U6b-BsaI-gRNA) digested with BsaI.
Tribolium DNA ligase 4
The Tribolium DNA ligase 4 ortholog (gene TC012219) was identified by
sequence similarity with Drosophila DNA ligase 4 protein, using BLAST.
A 2-kb fragment containing the largest exon was cloned from genomic
DNA, using primers AG001 and AG002 (supplementary material
Table S1), into pGEM-T-Easy (Promega). Double-stranded RNA was
produced as described previously (Posnien et al., 2009).
Tribolium injection and molecular analysis
Tribolium embryos were injected at the syncytial blastoderm stage and
handled as described previously (Berghammer et al., 2009). The injected
concentrations of Cas9, gRNA and knock-in templates are indicated in
Table 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 in
supplementary material Table S1), cloned and sequenced.
Acknowledgements
We thank Martin Klingler for discussions on the application of CRISPR in Tribolium;
Max Telford, Meriem Takarli and Josh Coulcher for preliminary experiments using
zinc-finger nucleases; Thomas Auer, Filippo del Bene and Marco Grillo for
discussing targeting strategies; and Gregor Bucher for sharing Tribolium stocks.
Competing interests
The authors declare no competing or financial interests.
Author contributions
A.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.
Funding
This work was supported by a PhD fellowship from the Université Claude Bernard
Lyon 1 (A.F.G.); a travel grant from the Boehringer Ingelheim Foundation (A.F.G.); a
post-doctoral fellowship of the Fondation pour la Recherche Mé dicale (J.B.S.); and
by a grant from the Agence Nationale de la Recherche [ANR-12-CHEX-0001-01].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.125054/-/DC1
2838
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