Zhang et al (2018) in a recent paper in PNAS report their successful integration of transgenes into the W chromosome of Bombyx mori. Initially, they set out to place visible markers on the W. This would enable rapid and accurate sexing of insects for sex-specific rearing of males for optimum cocoon production and controlled matings for egg production in the silk industry. Taking advantage of the transgenically marked W-chromosome, a second aim of this project was to develop a new approach for generating an early-acting female-lethal with the potential of being developed into a genetically-based SIT (gSIT) approach for controlling lepidopteran pests. The authors used a combination of TALENS and CRISPR/Cas9 transgenesis to achieve these goals.
In Lepidoptera, females are typically heterogametic, with a ZW or ZO chromosome constitution; by contrast, males are homogametic, designated ZZ. The W chromosome of the domesticated or mulberry silkworm, Bombyx mori, is among the best studied lepidopteran sex chromosomes. Heterochromatic staining makes it easy to differentiate from the autosomes, and as indicated by such staining properties, it consists mostly of transposable elements and other repeated sequences (Abe et al 2005).
The W also carries the so-called fem locus, a pi-RNA (piwi) producing sequence that interacts with the product of the Z-linked protein coding gene Masc, which is a central player in the early sex-determination cascade (Kawaoka et al. 2011) To localize the genetically defined fem locus on the W, Hiroaki Abe and colleagues mapped a dozen well-distributed W-specific RAPD sequences along the W-chromosome by classical genetic methods.
The first phase of Zhang et al’s project was to place one of two fluorescent markers into a unique RAPD site the Abe group had identified on the silkworm W-chromosome. For this Zhang et al. used TALENS technology, which remains one of the most effective transgenic techniques for silkworm. They designed plasmids with TALENS landing sites flanking a randomly chosen RAPD sequence, RIKISHI, containing a DsRed2 or GFP cassette driven by a baculovirus HR5-IE1 promoter.
The yield of transgenics was roughly comparable for both constructs: of 640 preblastoderm eggs injected in each case, 6 broods of G1 sib- or wild-type mated F1 individuals (from a total of 69 positive G0 individuals) produced offspring expressing DSRed2 (8.7%), and 2 broods (from 58 positives) expressed GFP (3.4%). The authors found high female-specific expression in both strains at all developmental stages from late embryogenesis on, and no apparent lack of fitness.
The second phase of the study involved construction of a two-part system to induce early female lethality using CRISPR-Cas9. To do this, they used the original TALENS induced W-linked GFP construct which also contained a cassette expressing Cas9 driven by the silkworm promoter for nanos (Bmnos), an essential early embryonic gene . Based on a previous study the authors then used piggyBac transformation to produce 3 independent autosomal lines carrying the strong ubiquitous silkworm U6 promoter driving a single guide RNA (U6 sgRNA) designed for transformer 2, (Bmtra2), an essential gene in the sex determination pathway they had shown acts as an embryonic lethal (Xu et al. 2017). Both transgenic lines produced progeny with wild type sex ratios and appeared normal phenotypically. Crossing females containing the W-Cas9 construct with homozygous U6 sgRNA-Bmtra-2 males produced normal numbers of F1 eggs; however, only males hatched from them, indicating virtually 100% expression of female lethality in early embryos.
Advantages of this study include the high efficiency of producing and screening for TALENs-rather than transposon-based (i.e., piggyBac) transgenics, the high, early expression of fluorescence and lethality in females, and the minimal number of strains needed to produce and maintain the dual CRISPR-Cas9 F1 hybrids (i.e., only one strain of females expressing Cas9 and another of males expressing an appropriate U6 sgRNA for the selected lethal gene). Thus this study represents an important step forward for sericulture and a promising pilot for future development of stable systems for gSIT-based control of lepidopteran and other insect pests with heterogametic sex chromosome consitution.