Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 RNPs

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economical impact and has become an emerging model for developing new genetic control strategies as alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific sgRNAs into early embryos. When targeting the eye pigmentation gene white eye (we), we observed a high rate of somatic mosaicism in surviving G0 adults. Germline transmission of mutated we alleles by G0 animals was on average above 70%, with individual cases achieving a transmission rate of nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in Ceratitis capitata will significantly advance the design and development of new effective strategies for pest control management.


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Nonetheless, a more comprehensive study of gene functions in Ceratitis will be needed to 75 further improve existing control strategies. To generate long-lasting and hereditable 76 changes in gene function, the CRISPR-Cas9 system with its modular and simple 77 components provides a promising tool to implement scalable, reproducible pest control 78 strategies 17,18 . Furthermore, transgene-based CRISPR-Cas9 can be used to produce 79 homozygous loss-of-function mutations as well as a novel gene drive system for insect 80 population control 19,21 .

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Altogether, we decided on the strategy of injecting Cas9 ribonucleoprotein (RNP) 93 complexes into insect embryos for the following reasons: 1) preloaded Cas9 RNPs should 94 act immediately following injection; 2) RNPs potentially result in higher efficiencies 95 compared to other approaches; 3) there are potentially less off-target events 30,31 ; and, 4) 96 Cas9 protein is more stable and robust than Cas9 mRNA, and can be generated with basic 97 protein purification strategies 30 .

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Here, we show that injecting in vitro generated Cas9-sgRNA RNPs into C. capitata 99 embryos is highly effective at inducing mono-and bi-allelic lesions of the targeted genes

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To test the feasibility of Cas9-mediated gene disruption in Ceratitis, we targeted the white 106 eye (we) gene, a locus required for eye pigmentation 4,5,32 . The Ceratitis we gene is the 107 ortholog of the X-linked white (w) gene in Drosophila 5 and is located on the Ceratitis 108 fifth chromosome 33,34 . The we gene is an ideal target to test Cas9-mediated disruption for Cas9 protein in Ceratitis embryos (Tab. 1). A slightly more pronounced toxicity of Cas9 118 was observed during transition from larvae to pupae (0.37.-0.49 compared to 0.84).

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We next loaded recombinant Cas9 protein with individual sgRNAs in vitro and injected 120 the RNP complexes into early syncytial embryos of the wildtype Benakeion strain. We 121 aimed at targeting syncytial nuclei to maximize the efficiency of inducing NHEJ lesions.

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A biallelic hit at this early stage is expected to produce large clones of mutant tissue in 123 injected individuals 22,30 . In this first round, we injected we-g1 RNPs in a buffer containing

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We performed additional rounds of embryos injections with we-g2 (150 mM KCl), and 138 we-g3 (300 mM KCl), and we subsequently observed either a lower percentage of adults 139 with eye pigmentation mosaicism (4% for we-g2) or none (we-g3). Injections of we-140 g1/we-g2 RNPs (150 mM KCl) led to 23% adult mosaics (Tab. 2), but this duplex RNP 141 targeting did not produce any deletions between the two targeted sites; furthermore, only 142 we-g2 induced indels were observed (reported in Fig. 1E, we-g2: 4-6). Again, sequencing 143 of cloned PCR products revealed NHEJ deletions, ranging from 2-21 bp, relative to the 144 PAM site of we-g2. 3 additional series injections of we-g1+ we-g2 into a total of 600 embryos (data not shown), also at 300 mM KCl, confirmed the lack of deletion between 146 the two targeted sites by PCR analysis on genomic DNA. The absence of a deletion 147 spanning the two targeted sites less than 100 bp apart can be explained, for example, by 148 steric hindrance which prevents that two adjacent Cas9-sgRNA complexes can cut both 149 sites simultaneously. Indeed, when injecting simultaneously we-g2 and we-g3 RNPs (in 150 300 mM KCl) in early embryos, we did recover deletions spanning the 2 more distant 151 targeted sites, ranging from 304 bp to 651 bp (Fig. 1F).

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Of the six we-g2 injected individuals, 4 males produced various proportions of G1 mutant 166 white-eyed progeny. Remarkably, the we-g1#1 and we-g2#5 lines gave rise to 100% and 167 98% G1 white-eyed offspring, respectively (Tab. 3). Thus, our results demonstrate that 168 Cas9 activity is highly effective in the germ line, producing mostly mutant primordial 169 germ cells.

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We randomly chose 4 out of 16 G1 we-g1-targeted mutant flies (two from we-g1#1 cross 171 and two from we-g1#2 cross) and 2 out of 166 we-g2-targeted mutant G1 flies (one from 172 we-g2#4 line and one from we-g2#5 line)(Tab. 3). Sequencing of cloned PCR products revealed that the 4 we-g1 G1 flies inherited one allele from the we parent and three 174 different we CRISPR alleles from the injected G0 fathers. We found an identical we deletion

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Approximately 90% of the embryos that failed to hatch (respectively, 90 embryos 221 and 160 embryos; Tab. 5) displayed disorganized cuticular structures. Some were up to 222 50% shorter in size compared to control embryos and consisted of a reduced number of 223 segments (Fig. 3 B). This phenotype is reminiscent of the pair rule phenotype described

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We report here that Cas9-mediated NJEH events generates lesions in the Ceratitis we and

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The reaction for complex formation was prepared by mixing 1.8 µg of purified Cas9

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The freshly prepared mixture was incubated for 10 min at 37°C and kept on ice prior to