Volume 32, Issue 6 p. 716-724
Original Article
Open Access

Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters

Rossina Novas

Rossina Novas

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina, USA

Pasteur+INIA Joint Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay

Contribution: Writing - original draft, ​Investigation, Methodology, Conceptualization, Data curation, Validation, Formal analysis

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Tatiana Basika

Tatiana Basika

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina, USA

Pasteur+INIA Joint Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay

Contribution: Writing - original draft, ​Investigation, Methodology, Conceptualization, Data curation, Validation, Formal analysis

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Megan E. Williamson

Megan E. Williamson

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina, USA

Contribution: ​Investigation, Methodology, Formal analysis

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Pablo Fresia

Pablo Fresia

Pasteur+INIA Joint Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay

Contribution: Writing - review & editing, Supervision, Funding acquisition

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Alejo Menchaca

Alejo Menchaca

Plataforma de Investigación en Salud Animal, Instituto Nacional de Investigación Agropecuaria (INIA), Montevideo, Uruguay

Contribution: Writing - review & editing, Supervision, Funding acquisition

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Maxwell J. Scott

Corresponding Author

Maxwell J. Scott

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, North Carolina, USA


Maxwell J. Scott, Department of Entomology and Plant Pathology, North Carolina State University, Campus Box 7613, Raleigh, NC 27695-7613, USA.

Email: [email protected]

Contribution: Writing - review & editing, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, ​Investigation, Formal analysis

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First published: 21 September 2023

Rossina Novas and Tatiana Basika contributed equally to this work.


The New World screwworm, Cochliomyia hominivorax, is an obligate parasite, which is a major pest of livestock. While the sterile insect technique was used very successfully to eradicate C. hominivorax from North and Central America, more cost-effective genetic methods will likely be needed in South America. The recent development of CRISPR/Cas9-based genetic approaches, such as homing gene drive, could provide a very efficient means for the suppression of C. hominivorax populations. One component of a drive system is the guide RNA(s) driven by a U6 gene promoter. Here, we have developed an in vivo assay to evaluate the activity of the promoters from seven C. hominivorax U6 genes. Embryos from the related blowfly Lucilia cuprina were injected with plasmid DNA containing a U6-promoter-guide RNA construct and a source of Cas9, either protein or plasmid DNA. Activity was assessed by the number of site-specific mutations in the targeted gene in hatched larvae. One promoter, Chom U6_b, showed the highest activity. These U6 gene promoters could be used to build CRISPR/Cas9-based genetic systems for the control of C. hominivorax.


Cochliomyia hominivorax, the New World screwworm (NWS) fly (Diptera: Calliphoridae), Coquerel 1858, is a primary myiasis-causing species that infests live animals during larval stages to feed on the host's flesh and fluids. Myiasis causes severe damage to livestock, generating great economic losses to the livestock industry by animal mortality and the cost of control mechanisms that are not efficient (i.e., insecticides, veterinary care, inspection). In Uruguay, economic losses, because of this pest, oscillate between USD 40 and 154 million annually (revised by Fresia et al., 2021).

NWS is endemic in South America and has been eradicated from North and Central America using the sterile insect technique (SIT; Knipling, 1959). To avoid reintroductions from the South, a barrier has been permanently maintained on the border of Panama–Colombia by releasing about 14 million sterile flies per week (Scott et al., 2017).

Genetic-based methods, such as CRISPR/Cas9-based systems, can expand the existing toolkit of NWS molecular methods, thus allowing the generation of more complex approaches for screwworm population control (Alphey & Bonsall, 2018; Scott et al., 2017). The CRISPR/Cas9 system consists of an endonuclease, Cas9, that produces a targeted double-strand break in a DNA sequence and a single-stranded RNA (gRNA) complementary to the DNA target sequence, that guides the endonuclease to the target site (Doudna & Charpentier, 2014). This double-stranded break can be repaired by the non-homologous end joining (NHEJ) mechanism, which commonly results in the introduction of deletions and/or insertions (collectively called indels) at the break site. Homology-directed repair (HDR) can also occur to repair the break, if supplied with a repair template that has a sequence complementary to either side of the damaged regions, incorporating this template in the genome (Champer et al., 2017).

The CRISPR/Cas9-based gene-editing system can be used to design a gene drive system. Gene drives are naturally occurring selfish elements that can increase the odds that they will be inherited. These elements serve as the basis for the development of ‘synthetic gene drives’ capable of spreading engineered traits through wild populations (Burt, 2003). Several synthetic Cas9-based gene drive systems, such as CleaveR (Oberhofer et al., 2019), toxin–antidote (Champer et al., 2020) and homing drive (Champer et al., 2018), have been developed and tested in the vinegar fly Drosophila melanogaster (Diptera, Drosophilidae), Meigen, 1830. Of these systems, the homing gene drive is attractive for population suppression of NWS. To build an autonomous CRISPR/Cas9-based homing gene drive, Cas9 and the guide RNA (gRNA) gene are inserted within the targeted gene at the location, which will be cut by Cas9/gRNA in the germline. Repair of the cleaved gene by HDR will lead to copying, or homing, of the Cas9 and gRNA transgenes into the targeted gene. If homing is efficient and not too costly to the organism, it will spread through susceptible wild populations (Esvelt et al., 2014).

We previously established the first protocol to generate site-specific modifications in the NWS fly genome using the CRISPR/Cas9 system, targeting and disrupting the transformer and brown body (yellow) genes (Paulo et al., 2019). In addition, the recently published whole-genome assembly of the NWS fly (Scott et al., 2020; Tandonnet et al., 2023) and other ongoing projects could allow the identification of other potential target genes, which could be used in future genetic control programs based on CRISPR/Cas9 (Fresia et al., 2021).

Building an efficient homing gene drive systems in NWS will require the identification of germline promoters for the expression of Cas9 and small RNA promoters for the expression of gRNA. In eukaryotes, small nuclear RNAs (snRNAs) are required for pre-mRNA splicing. Of these, U6 RNAs are synthesised by RNA polymerase III (Pol III; C. Li et al., 2004). The conserved features of U6 promoters have been thoroughly studied in D. melanogaster (Hernandez et al., 2007), as well as in other flies like Drosophila suzukii (Diptera, Drosophilidae), Matsumura, 1931 (Ni et al., 2021). In general, Pol III type 3 promoters, such as U6, consist of a ~21 bp proximal sequence element (PSEA) and a ~8 bp TATA box, which are located entirely upstream of the transcription start site (+1; Kim et al., 2020). The TATA box and the 5′ half of PSEA are well conserved but the 3′ half of PSEA varies among species. Previous research suggests that minor differences in the 3′ half of PSEA may contribute to RNA polymerase specificity (Hernandez et al., 2007). As a termination signal, Pol lll recognises stretches of T (Gao et al., 2018).

In this report, results from the in silico identification and in vivo validation of a set of U6 promoters for the expression of gRNA of C. hominivorax in a heterologous system are presented.


Identification of C. hominivorax U6 promoters

The U6 genes were identified in the screwworm genome by blast analysis of the C. hominivorax chromosome-scale genome assembly (Scott et al., 2020; Tandonnet et al., 2023) using D. melanogaster U6 genes as a query. U6 candidate genes were found in three clusters, all located in scaffold 470. Four U6 candidate genes were found in clusters 1 and 2, and five U6 candidates in cluster 3. A sequence analysis of the three clusters revealed that clusters 1 and 3 show extensive sequence similarity, but lower level of identity with cluster 2 (Figure 1a). Based on this sequence analysis, seven putative ChomU6 genes were identified and selected for further analysis: ChomU6_a, ChomU6_b, ChomU6_c from cluster 1, ChomU6_d_e and ChomU6_f from cluster 2 and ChomU6_g from cluster 3 (Figure 1a). Genomic coordinates of the ChomU6 gene candidates in the Scaffold 470 are shown in Table  1.

Details are in the caption following the image
The promoters from selected Cochliomyia hominivorax U6 genes. (a) Alignment of the ChomU6 gene clusters identified on the genome assembly. The green arrows indicate the U6 genes identified in each cluster, and the named ones are the genes whose promoters were selected for functional analysis. (b) Alignment of the six C. hominivorax U6 small nuclear RNA (snRNA) genes and their upstream regions. The three Drosophila melanogaster U6 genes and promoters are included in the alignment to highlight conserved elements. Coloured nucleotides indicate sequence identity. The proximal sequence element A (PSEA; −68 to −44), TATA box (−31 to −24), transcription initiation site (TIS; +1) and the U6 genes are underscored.
TABLE 1. Genomic coordinates (Tandonnet et al., 2023) of the Chom U6 genes and promoter regions.
U6 gene candidate Scaffold Coordinates (bp)
ChomU6_a 470 36,339,483…36,339,882
ChomU6_b 470 36,340,882…36,341,280
ChomU6_c 470 36,344,923…36,345,322
ChomU6_d_e 470 36,400,182…36,401,030
ChomU6_f 470 36,404,185…36,404,584
ChomU6_g 470 36,417,537…36,417,936

The predicted NWS U6 RNAs are very well conserved and present 100% sequence homology with the Drosophila U6 RNAs (Figure 1b). Upstream of the transcribed regions of the ChomU6 genes, we identified matches to the essential elements necessary for RNA Pol III recognition and binding motifs identified included a TATA box located at −31 to −24 bp from the predicted transcription initiation site (TIS), and the proximal sequence element (PSEA) found 68–44 bp upstream from the TIS. These elements showed some level of conservation with DmU6 genes regulatory elements (Figure 1b).

Validation of a gRNA targeting DsRed in a Lucilia cuprina transgenic line

With the aim of comparing the activities of ChomU6 gene promoters, we first sought to identify an efficient gRNA for expression in embryos. A gRNA for the endogenous gene yellow (Paulo et al., 2019) and two exogenous genes (DsRed and ZsGreen) were evaluated using both in vitro and in vivo assays. With the in vitro DNA cleavage assays, all gRNAs were effective at promoting Cas9-mediated cleavage of DNA fragments (Figure S1, Table S1). Consequently, we next evaluated the ability of Cas9/gRNA complexes to cleave the targeted gene using an in vivo assay. In our laboratory in North Carolina, we use the related blowfly Lucilia cuprina (Diptera, Calliphoridae), Wiedemann Pluetl 1830, as a model for evaluating genetic systems, which could be used for screwworm control (Edman et al., 2015; Yan et al., 2020). This is because C. hominivorax has been eradicated from North America and consequently we are not allowed to work with live C. hominivorax in our laboratory. The in vivo assay involved microinjection of precellular L. cuprina embryos with Cas9/gRNA complex and analysis of DNA extracted from developing G0 larvae for mutations in the targeted gene. The in vivo assays showed a range of indel rates, with some gRNAs being more active than others (Table S2). The final gRNA selected was gRNA-2 for the DsRed gene (gR-DsRed-2, Figure 2a), which produced the highest indel rate (Table S2).

Details are in the caption following the image
Validation of a DsRed gene as a potential CRISPR/Cas9 target in Lucilia cuprina embryos. (a) Schematic of the Cas9-targeted regions in the DsRed coding region and the sgRNAs tested. Targeted sites are indicated by a scissor, PAM motif in black and amplification primers as dark red arrows. Fragment size patterns expected after in vitro Cas9 activity assay are indicated for each gRNA. (b) Agarose gel analysis of the in vitro cleavage products after incubation of the polymerase chain reaction (PCR) amplification product with Cas9/gRNA complex. (c) Schematic of the in vivo assay used to evaluate DsRed gRNA activity in L. cuprina embryos. (d) CRISPResso indel analysis. Left, editing frequency of reads as determined by the percentage of sequence reads showing unmodified and modified alleles. Right, visualisation of the distribution of identified alleles around the cleavage site for the gR-DsRed2. No cutting activity was observed when injecting gR-DsRed1 (Table S2).

The workflow for screening and analysing the editing efficiency is indicated in Figure 2c. The individual in vitro transcribed gRNA (200 ng/μL) was mixed with a high concentration of Cas9 protein (750 ng/μL) to form ribonucleoprotein complexes (RNPs). Preassembled RNPs were coinjected with 300 ng/μL of a ZsGreen fluorescent protein expressing plasmid (Concha et al., 2011) into 173 early pre-blastoderm L. cuprina EF3E strain embryos, which has the DsRed gene integrated into the genome as a transgene (Yan et al., 2020). In total, 72 ZsGreen positive L1 larvae were recovered (hatching rate: 42%), while control (non-injected) embryos displayed a hatching rate of 72% (30 L1 recovered from 42 embryos). Insertion and deletion (indel) evaluation by amplicon sequencing followed by CRISPResso2 software analysis revealed a 78% mutagenesis rate. A diversity of mutated alleles was recovered, including small and medium deletions (between 1 and 10 bp) and some insertions (Figure 2d). All the mutations were within the region of the DsRed gene targeted by gR-DsRed2, showing this is an efficient gRNA to move forward with for testing ChomU6 promoters.

In vivo testing of ChomU6 promoters in L. cuprina

To test the efficiency of the predicted ChomU6 promoters, the regions upstream of each selected U6 candidate gene (including the regulatory elements) and the terminator regions were synthesised and cloned into pUCIDT vectors. In the case of the ChomU6_d_e double promoter, gR-DsRed-2 was inserted downstream of both promoters, so we cannot distinguish if the activity is due to one promoter, the other or the combination of the two. However, this double promoter is of interest as it could potentially be used to express two different gRNAs from a single gene construct and may increase editing efficiencies (Port et al., 2014). Recognition sequences of Type IIS restriction enzymes for Golden Gate cloning were included to facilitate the insertion of the gR-DsRed2 sequence, following a similar strategy that was used to express gRNAs from U6 promoters in D. melanogaster (Port et al., 2014).

The activity of each ChomU6-gRNA construct was tested using an in vivo assay. The workflow for the assay is indicated in Figure 3a and is similar to that described above for testing gRNAs with Cas9 protein. Each ChomU6 construct (500 ng/μL) was tested in two different conditions: First, co-injecting with Cas9 purified protein (750 ng/μL) and second, with a plasmid that expresses Cas9 from the constitutive Chomhsp83 gene promoter (500 ng/μL). For both conditions, a plasmid expressing the fluorescent marker ZsGreen (300 ng/μL) was included in the microinjection mix. Approximately 250 L. cuprina syncytial embryos were injected with each plasmid DNA or plasmid/Cas9 protein mix. Next, larvae that developed from injected embryos and expressed the fluorescent marker were collected, DNA isolated and PCR performed with DsRed primer pairs (Figure 2a). The amplification products were analysed via deep sequencing using the CRISPResso2 tool.

Details are in the caption following the image
Cochliomyia hominivorax U6 gene promoters are active in Lucilia cuprina embryos. (a) Schematic workflow of the in vivo assay used to evaluate ChomU6 promoters. (b) Summary of the results obtained from the in vivo analysis. Each ChomU6-gRNA plasmid was co-injected with either Cas9 protein or a plasmid that expresses Cas9. See Table S3 and experimental procedures for additional information on allele editing % (indel %) calculations.

Varying degrees of in vivo activity for each ChomU6 promoter were observed in L. cuprina, as reflected by the editing rates at the DsRed gene (Figure 3b, Table S3). However, the level of editing observed was similar with the two different sources of Cas9 for each ChomU6 promoter evaluated. ChomU6_a, c and g showed no editing activity compared to control uninjected embryos (Table S3). Of the three ChomU6 promoters that showed activity, ChomU6_b had the highest editing rates (1.17% with co-injected Cas9 plasmid and 0.68% with co-injected Cas9 protein).


With precisely defined transcription initiation and termination sites, the RNA Pol III promoters and terminators from U6 genes are advantageous for the expression of gRNAs in transgenic insects. Consequently, U6 gene promoters have been isolated and characterised by several insect species (Feng et al., 2021; Huang et al., 2017; Ni et al., 2021; Port et al., 2014). In this report, we tested the in vivo activity of seven C. hominivorax U6 promoters. All promoters contained the conserved PSEA and TATA elements characteristic of U6 promoters (Figure 1). However, the promoters varied in activity with three showing little activity above background. It is not obvious from an examination of the PSEA and TATA elements why some promoters are more active than others. Indeed, the TATA element for the most active promoter ChomU6_b, 5′-TACAAATA-3′ is not a particularly good match to the consensus (Lobo & Hernandez, 1989). However, a variation in activity among ChomU6 promoters was not unexpected as, in other insects, U6 promoters differ substantially in the ability to express gRNAs such as in D. melanogaster (Port et al., 2014), D. suzukii (Ni et al., 2021), Plutella xylostella (Lepidoptera, Plutellidae), Linnaeus 1758, (Huang et al., 2017), among others (Feng et al., 2021).

Three clusters of U6 genes were identified on one of the chromosome-length scaffolds in the current genome assembly. Clusters 1 and 3 showed extensive nucleotide similarity. It would appear that these clusters are the same but were misassembled onto separate regions of the scaffold but with an additional U6 gene at the 3′ end of cluster 3. We noted previously that residual heterozygosity in the inbred strain appears to have led to divergent alleles of some regions of the genome to be assembled onto separate contigs (Scott et al., 2020; Tandonnet et al., 2023).

For the in vivo U6 promoter assay, we injected embryos from the blowfly L. cuprina since C. hominivorax has been eradicated from North America, and consequently, we are not allowed to work with this species in our laboratory in North Carolina. Both C. hominivorax and L. cuprina belong to the family Calliphoridae but have different subfamilies: Luciliinae (Lucilia) and Chrysominae (Cochliomyia; Nelson et al., 2012). We have previously found that Pol II Cochliomyia gene promoters are active in L. cuprina (Edman et al., 2015). Consequently, we expect that ChomU6 promoters found to be active in L. cuprina will also be active in C. hominivorax but this remains to be shown. Further, it is possible that the most active promoter identified in this study, ChomU6_b, may not be the most active U6 promoter in C. hominivorax embryos. A biosecure facility for screwworm research is under construction in Uruguay for testing U6 promoters and other essential components for a population suppression gene drive.

The in vivo assay with ChomU6 promoters showed relatively low editing efficiency compared with injections with the same gRNA in preformed RNP complexes. Perhaps the time taken for the gRNA to be transcribed from the ChomU6 plasmids sufficiently delayed the assembly of functional Cas9/gRNA complexes to reduce the amount of editing in L. cuprina embryos, which develop rapidly at room temperature. While several studies report high editing efficiency with gRNAs expressed from U6 promoters on plasmid DNAs (Ahmed et al., 2019), other investigations found editing efficiencies similar to this study (Ni et al., 2021). For example, in the mosquito Culex quinquefasciatus (Diptera, Cullicidae), Say 1823, (Feng et al., 2021), the highest editing efficiency was around 1.5% for a gRNA expressed from the U6:1 promoter.

The endogenous ChomU6 promoters identified in this study, particularly the ones that show highest activity, will be valuable for the efficient expression of gRNAs in gene-editing strategies. In the future, evaluation of the activity of these promoters in C. hominivorax and the generation of lines expressing these constructs will aid towards the development of genetic tools for pest management such as gene drives for population suppression (Esvelt et al., 2014; Kyrou et al., 2018).


Insect rearing

The LA07 WT strain of L. cuprina was maintained as previously described (F. Li et al., 2014; Yan et al., 2020). Adults were kept in mesh cages at 22°C and fed a sugar/water/protein biscuit diet. Larvae were raised on 93% ground beef at 27°C, and pupae were kept in a 27°C incubator until eclosion.

gRNA design and synthesis

Single gRNAs were designed as previously described (Paulo et al., 2019, 2022). Briefly, target exons sequences were examined for the presence of protospacer-adjacent motifs (PAMs, sequence NGG-3′, where ‘N’ is any base) using the standalone version of CRISPOR tool (Concordet & Haeussler, 2018) in the context of C. hominivorax genome (GenBank: GCA_004302925.1; Tandonnet et al., 2023).

gRNAs were synthesised by first generating templates by PCR in 100 μL final volume containing 1× Q5 High-Fidelity 2× Master Mix (New England Biolabs, NEB) and 0.5 M of each CRISPR primer (Paulo et al., 2022). Amplification conditions included an initial denaturation step of 98°C for 2 min, followed by 35 cycles of 98°C for 10 s, 58°C for 10 s and 72°C for 10 s, followed by a final extension of 72°C for 7 min. The expected 100 bp amplicons were confirmed by loading 5% of the PCR reaction on a 1× Tris-Borate-EDTA (TBE) buffer 1% agarose gel and run at 90 V for 90 min. The remaining PCR product was purified using the QIAquick PCR Purification Kit (Qiagen) and quantified with the Qubit HS-DNA Kit (Thermo). In vitro transcription of the gRNAs was carried out using the MEGAshortscript T7 Transcription Kit (Thermo) and 300 ng of DNA template in a final volume of 20 μL. Reactions were incubated at 37°C overnight followed by TURBO DNase (Thermo) treatment using 2 U for a further 15 min at 37°C. Transcriptions were extracted with Phenol: Chloroform: Isoamyl Alcohol (25:24:1 v/v, pH 6.7), precipitated with isopropanol and left overnight at −20°C. RNA was collected by centrifugation, washed once with 75% ethanol and resuspended with RNAse-free water. Concentrations of the gRNAs were measured using Qubit (Thermo), aliquoted and stored at −80°C until use.

Cas9 in vitro cleavage assay

In order to determine the cleavage efficiency of the gRNAs designed against each selected target, an in vitro cleavage assay using Cas9 (TrueCut™, Thermo Fisher) was performed, as previously described (Paulo et al., 2022) with some modifications. Briefly, a template containing the target site was amplified from 50 to 100 ng of genomic DNA using Q5® High-Fidelity 2× Master Mix (NEB), following manufacturer instructions. Cas9 and gRNA were complexed in a reaction containing 600 ng of sgRNA, 0.7 μL of Cas9 (30 μM) and nuclease-free water to a final volume of 27 μL, incubating the mix 10 min at 37°C.

Then, 100 ng of template DNA was added for a final volume of 30 μL. Samples were incubated at 37°C for 1 h and then resolved in 2% agarose gels.

Plasmid constructions

For each individual C. hominivorax U6 promoter identified in the in silico analysis, approximately 500 bp upstream the TIS were selected, and the following 5′-to-3′ configuration was synthesised and cloned into a pUCIDT vector (IDT): ChomU6 promoter followed by a restriction site linker with two BbsI sites, the tracRNA (RNA core) sequence from DmU6:3-3′UTR sequence (Port et al., 2014) and the U6 gene 3′ UTR. The double promoter was cloned into pUC57-mini vector (GenScript), following the same configuration, with two BbsI sites for ChomU6_d and two BsmBI sites for ChomU6_e. The gRNAs target sequence was inserted using Golden Gate assembly, using the sense and antisense oligos listed in Table S4, and following a similar strategy as for Drosophila U6 promoters (Port et al., 2014). The plasmids were transformed into 10-beta Competent Escherichia coli (NEB) (Enterobacterales, Enterobacteriacaea), Castellari and Chalmers, 1919. Correct clones were subsequently identified by restriction digestion using PspOMI and XhoI (NEB) and confirmed by performing Sanger sequencing with specific primers for each promoter. Plasmid DNA was purified from the correct colonies and prepared for injections.

For the generation of the pBS-Chomhsp83-NLS-SpCas9-NLS plasmid, first, the pBS-Dmhsp70-Cas9 plasmid (Addgene Plasmid #46294) was digested with ClaI and ends blunted using Klenow polymerase (NEB). The linear plasmid was then PCR purified and digested with XhoI and treated with Antarctic phosphatase (NEB). The Chomhsp83 promoter was isolated from the plasmid DR4 (Linger et al., 2015) using the Chomhsp83_for and Chomhsp83_rev primer set with a SmaI site added to the 5′ end of the forward primer and a XhoI site added to the 5′ end of the reverse primer. This fragment was amplified, and the PCR product was then digested with SmaI and XhoI and ligated to pBS-Dmhsp70-Cas9. The ligation products were transformed into 10-beta Competent E. coli (NEB). Colonies were confirmed by restriction digest and Sanger sequencing. Plasmid DNA was purified from the correct colonies and prepared for injections.

All primers used are listed in Table S4.

Embryo microinjections

RNPs were preassembled by incubating Cas9 protein (750 ng/μL) with specific gRNA (200 ng/μL) in a sodium phosphate buffer (supplemented with 300 mM of KCl) at 37°C for 30 min. The plasmid pB[Lchsp83-ZsGreen] (Concha et al., 2011) was added to the final injection cocktail (300 ng/μL). The injected DNA plasmids were prepared using ZymoPURE II Plasmid Midiprep Kit. To prevent needle clogging, the injection cocktail was spun through a Ultrafree-MC HV Centrifugal Filter (Millipore) for 4 min at 12,000g, and the mix was maintained on ice during the experiments. Needles were prepared with a P-2000 needle puller (Sutter Instrument) using quartz capillaries with filament (O.D.: 1.0 mm; I.D.: 0.7, 10 cm length) and bevelled using a BV-10 Micropipette Beveler (Sutter Instrument). Embryos were aligned on a double slide tape in concave well microscopy slides, dehydrated in a silica gel chamber for 6 min 30 s, and then covered with Halocarbon 27 oil (Sigma).

Microinjections were performed at the posterior end of pre-blastoderm L. cuprina embryos within the first 45 min of embryogenesis. Injections were performed using a XenoWorks micromanipulator connected to a digital microinjector (Sutter Instrument) device set for a ‘continuous’ injection mode. After microinjection, the slides were placed into a hyperbaric oxygen chamber and incubated overnight at 21–25°C. Eclosing first instar G0 larvae showing fluorescent marker expression were identified using a Leica M165FC stereomicroscope and collected in a 1.5 mL tube maintained at −80°C until processing for genomic DNA extraction.


Genomic DNA was extracted using the DNA Miniprep Plus Kit (Zymo). PCR reactions were carried out for a final volume of 50 μL containing 1× Q5 High-Fidelity 2× Master Mix (NEB), 0.5 μM of each target-specific forward and reverse primers (Table S4). Samples were cycled under the following conditions: 98°C for 30 s, 35 cycles of 98°C for 10 s, 60°C for 30 s and 72°C for 30 s, followed by a final extension step at 72°Cfor 2 min. PCR products were verified by electrophoresis in a 1% agarose gel and purified using DNA Clean & ConcentratorTM-5 Kit (Zymo). Purified PCR products were submitted for next-generation sequencing to Genewiz, and results were analysed by CRISPResso2 (Clement et al., 2019). Allele editing percentage was calculated as: allele editing% (# of reads with modified alleles excluding alleles modified by only substitutions)/(# of total reads aligned to target) (Table S3). We did not include mutated alleles modified by only substitutions, as these are mostly derived from recurring single-nucleotide polymorphisms and amplification/sequencing artefacts. Instead, we analyse indels (deletions and insertions), which are a robust signature of cellular repair mechanisms resulting from Cas9 nuclease activity (Feng et al., 2021).


Rossina Novas: Writing – original draft; investigation; methodology; conceptualization; data curation; validation; formal analysis. Tatiana Basika: Writing – original draft; investigation; methodology; conceptualization; data curation; validation; formal analysis. Megan E. Williamson: Investigation; methodology; formal analysis. Pablo Fresia: Writing – review and editing; supervision; funding acquisition. Alejo Menchaca: Writing – review and editing; supervision; funding acquisition. Maxwell J. Scott: Writing – review and editing; conceptualization; funding acquisition; project administration; resources; supervision; investigation; formal analysis.


We thank Esther Belikoff for training on embryo microinjections and blowfly rearing and our colleagues in the Scott lab for helpful discussions.


    This research was supported by an agreement between The Institut Pasteur de Montevideo and North Carolina State University and grants from the Inter-American Development Bank (IBD UR-T1227) and from INIA (FTPA N°359). Tatiana Basika, Rossina Novas, Pablo Fresia and Alejo Menchaca are members of SNI (National Research System, Uruguay).


    The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


    The datasets generated for this study can be found in the manuscript and the Supplementary Material. The Genbank accession numbers for the plasmids described in this paper are OQ434249–OQ434255.