New Results

Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in the domesticated silkworm, Bombyx mori

Kenta Tomihara, Peter Andolfatto, Takashi Kiuchi
doi: https://doi.org/10.1101/2022.05.07.490996

Abstract

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

Brief abstract Bombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene”s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori × B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication.

Introduction

The silkworm, Bombyx mori, was domesticated over 5000 years ago from its wild progenitor species, B. mandarina. Under long-term artificial selection, B. mori acquired various characteristics suitable for sericulture. For example, the weight of the cocoon shell of B. mori is much higher than that of B. mandarina (Ômura 1950, Li et al. 2017, Fang et al. 2020). In addition, B. mori moths lost their flying ability due to the degeneration of their flight muscles and a reduction in wing stiffness (Lu et al. 2020). Among the most conspicuous domestication-associated traits is a marked reduction in pigmentation in B. mori larvae and adults relative to B. mandarina (Figure 1A). Curiously, depigmentation is a major trait contributing to the so-called “domestication syndrome” observed in a variety of domesticated animals, but for reasons that are not well-understood (Wilkins et al. 2014).

Figure 1.
Figure 1.

(A) Larvae (top) and adult males (bottom) of B. mori (left) and B. mandarina (right). Black arrows indicate larval spot markings (eye spots, crescent spots and star spots) and red dotted lines delimit the larval dorsal pigment patterning (“banding”). The names of spots follow the nomenclature of Yoda et al. (2014). (B) The proposed melanin biosynthesis pathway in Bombyx larvae largely adapted from Futahashi et al. (2008) and Yoda et al. (2014). Orange dashed arrows indicate presumed regulation of genes by the transcription factor apt-like.

The genetic basis of pigmentation loss associated with B. mori domestication is not yet known. Previous studies reported that melanin synthesis pathway genes tyrosine hydroxylase (TH) and aspartate decarboxylase (ADC, also known as black) were potentially targets of selection during silkworm domestication (Yu et al. 2011, Xiang et al. 2018). Pigmentation patterning genes are also likely to be associated with the body color differences between B. mori and B. mandarina. The wild-type B. mori larvae are largely white but exhibit melanic spots (i.e. eye spots, crescent spots and star spots, Figure 1A). B. mandarina larvae, on the other hand, are substantially darker, and exhibit extensive dorsal pigment patterning that includes banding and spots (Figure 1A).

In B. mori, distinct alleles of the genetic locus, p, encode at least 15 different larval markings such as spots, stripes, and banding (Yoda et al. 2014). The gene underlying allelic variation at p, apontic-like (apt-like), encodes a transcription factor that is likely to regulate the expression of melanin synthesis pathway genes such as yellow-y, ebony, TH, Dopa decarboxylase (DDC) and laccase 2 (Futahashi et al. 2008, Yoda et al. 2014) (Figure 1B). While apt-like has been implicated in pigmentation differences among B. mori strains, it”s potential role in depigmentation of B. mori during domestication is not known. A hybrid strain (semiconsomic T02), in which chromosome 2 of B. mandarina has been substituted into the genomic background of B. mori, exhibits a phenotype similar to that of the B. mori allele moricaud (pM) (Fujii et al. 2021). Since apt-like resides on chromosome 2, dorsal pigmentation patterning on the larvae of B. mandarina that is absent in B. mori was hypothesized to be controlled by the expression of apt-like (Yoda et al. 2014, Fujii et al. 2021), although direct evidence is lacking.

The genetic analysis of domestication-associated loss of pigmentation in B. mori has been challenging because deficiencies in candidate genes can result in lethality. For example, in the fruit fly, Drosophila melanogaster, TH-deficient (pale) mutants die at embryonic stage (Neckameyer and White 1993). In B. mori, TH-deficient mutants (sch lethal, schl) and RNAi-mediated knockdowns of TH are both lethal at embryonic stage (Liu et al. 2010). In addition, apt mutants in D. melanogaster die at embryonic stage (Eulenberg and Schuh 1997, Gellon et al. 1997), and RNAi-mediated knockdown of apt-like in B. mori embryos results in death before hatching (Yoda et al. 2014).

The issue of lethality is likely to continue to impede the genetic analysis of pigmentation loss and other domestication-associated traits in B. mori. Recently, several candidate genes associated with domestication in B. mori have been identified using quantitative trait locus (QTL) mapping, including silk production (Li et al. 2017, Fang et al. 2020), larval climbing ability (Wang, Lin, et al. 2020), and mimicry (Wang, Lin, et al. 2020). In addition, a recent population genetic analysis identified 300 candidate genes as targets of recent selection in B. mori, some of which are likely to be associated with silk production and voltinism (Xiang et al. 2018). Despite these efforts, reverse genetic approaches such as targeted gene knockouts and editing (Takasu et al. 2010, 2013, Wang et al. 2013), gene silencing (Quan et al. 2002), or transgenesis (Tamura et al. 2000) will likely be required to fully understand the function of these candidate genes and their potential contribution to domestication-associated traits in B. mori. Notably, the functions of domestication candidate genes have been tested by reverse genetics approaches in B. mori (Xiang et al. 2018) but these approaches, to our knowledge, have not yet been implemented in B. mandarina.

Here we use CRISPR/Cas9-targeted knockouts of two candidate pigmentation genes in two distinct contexts. First, we demonstrate the feasibility of CRISPR/Cas9-targeted knockouts in B. mandarina by generating a homozygous yellow-y knockout strain. Next, to circumvent the lethal effects of knocking out a second candidate gene, apt-like, we use allele-specific CRISPR/Cas9-targeted knockouts in B. mori × B. mandarina F1 hybrids. The latter experiment also comprises a test (the “reciprocal hemizygosity test”, Steinmetz et al. 2002, Stern 2014) of the contribution of apt-like evolution to the domestication-associated loss of pigmentation in B. mori.

Results and discussion

A CRISPR/Cas9-targeted knockout of yellow-y in B. mandarina

To demonstrate the feasibility of CRISPR/Cas9-targeted knockouts in B. mandarina, we focused on known pigmentation-related genes. TH is among the most compelling candidate genes in the melanin synthesis pathway that seems likely to underlie domestication-associated loss of pigmentation in B. mori (Yu et al. 2011, Xiang et al. 2018). However, TH knockout mutants are predicted to be lethal (Neckameyer and White 1993, Liu et al. 2010), rendering them difficult to study. Thus, we decided to target yellow-y (Figure 1B), a melanin synthesis gene that is downstream of TH and functions in melanin synthesis in wide range of insects including B. mori and other Lepidoptera and is predicted to be non-essential (Futahashi et al. 2008, Zhang et al. 2017, Chen et al. 2018, Matsuoka and Monteiro 2018, Liu et al. 2020, Wang, Huang, et al. 2020, Han et al. 2021, Shirai et al. 2021). After confirming the coding sequence annotation (CDS) of the B. mandarina yellow-y gene, we designed a unique CRISPR-RNA (crRNA) target site in exon 2 (Figure 2A, Table S1).

View this table:
Table S1.

PCR primers and RNA sequences used in this study.

Figure 2.
Figure 2.

The nature of the lesion in the yellow-y knockout (yellow-yΔ15) in B. mandarina.

(A)Gene structure of yellow-y and the selected crRNA target site. The target sequence is underlined and the protospacer adjacent motif (PAM) site is shown in bold letters. (B) Alignment of the yellow-y gene sequences surrounding the crRNA target site from wild type, and G2 B. mandarina individuals that are homozygous for a 5-base pair deletion and associated single nucleotide mutation. The target sequence is underlined, and the PAM site (CCN) is indicated in bold letters.

In B. mori, CRISPR/Cas9-targeted genome editing requires microinjection into non-diapausing eggs (Kanda and Tamura 1994). We obtained non-diapausing eggs by rearing B. mandarina larvae under 16 h-light/8 h-dark conditions (Kobayashi 1990). We then injected a mixture of crRNA, trans-activating crRNA (tracrRNA), and Cas9 into 336 B. mandarina embryos. Among 16 hatched larvae (G0 generation), nine grew to adult moths (Table 1). We crossed six G0 adults with wild-type moths and obtained generation 1 (G1) eggs. Using a heteroduplex mobility assay on the PCR products from G1 embryos, we confirmed that mutations were introduced at the target site in five of the six G1 broods (Table 1, Figure S1), showing that CRISPR/Cas9-induced mutations of yellow-y were heritable. We then crossed G1 siblings with each other and obtained yellow-y homozygous knockout individuals carrying a five-nucleotide deletion followed by a single-nucleotide substitution (Figure 2B), which results in a frame-shift and premature stop codon. We designated this mutant allele yellow-yΔ5 and used it for further analyses.

Figure S1.
Figure S1.

Detection of mutations at the yellow-y crRNA target site using a heteroduplex mobility shift assay. Two intervals were prepared for each G1 brood. Ten eggs were collected into one tube, and genomic DNA was prepared. The region containing the target site of yellow-y crRNA was PCR-amplified. Multiple heteroduplex bands caused by insertion/deletion mismatches were observed indicating the presence of DNA lesions (insertions/deletions and/or associated nucleotide variants).

View this table:
Table 1.

Efficiency of CRISPR/Cas9-targeted knockout in B. mandarina targeting yellow-y.

B. mandarina yellow-yΔ5 homozygotes hatched normally and their development was comparable to that of wild-type individuals (Figure 3). In homozygous yellow-yΔ5 neonate larvae, the larval integument and the head capsule are reddish brown instead of the normal black (Figure 3A) and in final instar larvae, spots and dorsal pigmentation patterns are lighter than that of the wild type (Figure 3B). Later in development, the pupal integument of homozygous yellow-yΔ5 mutants exhibits reddish color instead of the normal black (Figure 3C), and the body and wing spot markings of yellow-yΔ5 adult moths are lighter than that of wild-type (Figure 3D). Further, the phenotypes of heterozygous +/yellow-yΔ5 individuals were comparable to that of wild type (data not shown). Together, these observations suggest that, as observed in B. mori (Futahashi et al. 2008), yellow-y is a non-essential gene contributing to melanin pigment synthesis in B. mandarina and loss-of-function is recessive.

Figure 3.
Figure 3.

The phenotypes of wild-type and yellow-y knockout mutants of B. mandarina. Representative (A) neonate larvae, (B) final-instar larvae, (C) male pupae and (D) adult male moths of wild-type (left or top) and yellow-yΔ5 (right or bottom) B. mandarina. Arrows indicate wing spot markings in adult moths. Scale bars: 1 mm (A) or 1 cm (B-D).

Comparative data from other species suggests that yellow-y functions differently among lepidopteran species. For example, while yellow-y loss-of-function is also found to be recessive in several other lepidopteran species (Liu et al. 2020, Wang, Huang, et al. 2020, Han et al. 2021, Shirai et al. 2021), it is dominant in the black cutworm, Agrotis ipsilon (Chen et al. 2018). Further, unlike Bombyx, yellow-y knockouts in Agrotis are susceptible to dehydration (Chen et al. 2018) and mutants in Spodoptera exhibit defects in body development, copulation, oviposition, and hatchability (Liu et al. 2020, Han et al. 2021, Shirai et al. 2021). These observations suggest that although the function of yellow-y in melanin synthesis is conserved, additional yellow-y functions might be diverged among lepidopteran species. yellow genes are a rapidly evolving gene family, and loss or duplication of some yellow genes have been observed in Lepidoptera (Chen et al. 2018, Liu et al. 2020, Han et al. 2021, Shirai et al. 2021). It is possible that functions of yellow-y outside of melanin synthesis in Bombyx are compensated for by other yellow paralogs. In Lepidoptera, the function of most yellow genes remained unknown except for yellow-y (Futahashi et al. 2008, Zhang et al. 2017, Chen et al. 2018, Matsuoka and Monteiro 2018, Liu et al. 2020, Wang, Huang, et al. 2020, Han et al. 2021, Shirai et al. 2021), yellow-e (Ito et al. 2010), yellow-d (Zhang et al. 2017) and yellow-h2/3 (Zhang et al. 2017). Further studies of yellow genes including yellow-y in diverse insect species are required to understand the functions and evolution of yellow paralogs.

While our results demonstrate the feasibility of genome editing in B. mandarina, these experiments are challenging due to low post-injection hatchability rates (4.8% in our experiment). A recent study showed that the hatchability of B. mandarina eggs is generally lower than B. mori eggs, even under normal conditions (Zhu et al. 2019). To compare the hatchability of B. mori and B. mandarina embryos after injection, we injected three commonly used buffers (see Methods) and distilled water into embryos of both species and compared hatching rates. We found that the post-injection hatchability of B. mandarina embryos (0–8.3 %) is substantially lower than that of B. mori (22.9–62.5 %) in all experimental conditions (Table 2), suggesting that B. mandarina embryos are more sensitive to injection.

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Table 2.

The hatchability of B. mori and B. mandarina embryos injected with an injection buffer 1 (Yamaguchi et al. 2011), injection buffer 2 (Tamura et al. 2000), PBS buffer and distilled water.

Allele-specific knockouts of apt-like in interspecific F1 hybrids

Knockouts of some pigmentation pathway genes, such as apt-like and TH, are predicted to be lethal (Neckameyer and White 1993, Eulenberg and Schuh 1997, Gellon et al. 1997, Liu et al. 2010, Yoda et al. 2014). Considering this obstacle to the study of essential genes, together with the observation (above) of reduced hatchability of injected B. mandarina embryos, we instead opted for the alternative strategy of injecting B. mori × B. mandarina F1 hybrids. Specifically, we conducted an allele-specific CRISPR/Cas9-targeted knockout of apt-like in F1 embryos (from crosses between B. mori females and B. mandarina males). We reasoned that, because the components of these F1 eggs is derived from the B. mori mother, these embryos should have post-injection hatchability similar to that of B. mori. We designed species-specific crRNA targeting apt-like, for which the targeted protospacer adjacent motif (PAM) sequence is only present in either B. mori- or B. mandarina-derived sequence (Methods, Figure 4, Table S1). For each targeted allele, we injected a mixture of crRNA, tracrRNA and Cas9 into 48 F1 embryos. To confirm that mutations were specifically introduced into the targeted allele, we extracted genomic DNA from adult legs and PCR-amplified the target sites. We then carried out heteroduplex mobility assays using a microchip electrophoresis system (Ota et al. 2013, Ansai et al. 2014), which show that various mutations were introduced into the apt-like target sequence in both of the allele-specific knockout series (Figure S2). The PCR products obtained from two representative individuals of each allele-specific knockout series were then cloned and sequenced (Figure 4). The sequences of cloned PCR products confirmed that various mutations were introduced into the target sites in both knockout series, some of which cause frameshifts and associated premature stop codons. All mutations detected in this experiment were specifically introduced only into the targeted allele.

Figure S2.
Figure S2.

Detection of mutations at apt-like crRNA target sites using a heteroduplex mobility shift assay. The region containing the target site of (A) B. mori specific apt-like crRNA and (B) B. mandarina specific apt-like crRNA was PCR-amplified using DNA prepared from G0 adults” legs. Multiple heteroduplex bands caused by insertion/deletion mismatches and associated nucleotide variants were observed in G0 mosaics.

Figure 4.
Figure 4.

Mutations introduced by allele-specific apt-like mosaic knockout (mKO) in F1 hybrids. Annotation and inset alignments of the apt-like gene in both species with selected crRNA target sites whose PAM sequences are only present in (A) B. mori or (A) B. mandarina. Two allele-specific knockout individuals were sequenced for each target. The target sequences are underlined, and the PAM sequences are shown in bold letters. B. mori specific SNPs and B. mandarina specific single nucleotide variants are highlighted with green and yellow shadings, respectively. Mutations introduced by CRISPR/Cas9 system are highlighted with red shading. Numbers on the right edge indicate the numbers of the clones identified among all cloned and sequenced PCR products.

In addition to spots, normal F1 larvae have a dark body with darker greyish brown banding covering wide range of the dorsal surface, similar to the pattern for B. mandarina (Figure 1A, Figure S3). As predicted, post-injection hatchability of F1 embryos was high whether targeting the B. mori (60%) or the B. mandarina (66%) allele. In the apt-like knockout series targeting the B. mori allele, all of the 26 larvae that survived to the fifth instar stage exhibited normal body color (Table 3, Figure 5). In contrast, for the apt-like knockout series targeting the B. mandarina allele, 24 of the 29 larvae that survived to the fifth instar stage exhibited white patches on dorsal pigmentation pattern banding that varied in size (Table 3, Figure 5, Figure S4). This result establishes a role for apt-like in body pigment formation and that the B. mandarina-derived apt-like allele is dominant with respect to this trait. Notably, however, the pigmentation of F1 pupae and adult stages of the knockout series targeting the B. mori or the B. mandarina allele both exhibited normal body color (data not shown). Additionally, larval spots, which are also predicted to be controlled by apt-like (Yoda et al. 2014), were also not affected the F1 series targeting either the B. mori or the B. mandarina allele (Figure 5, Figure S4). Since both wild-type (+p) B. mori and B. mandarina larvae exhibit spots (Figure 1A), we conclude that the B. mori or the B. mandarina-derived apt-like alleles are both sufficient to direct the formation of larval spots.

Figure S3.
Figure S3.

A representative (A) fifth instar larva and (B) adult male moth of normal F1 (B mori female × B. mandarina male). Black arrows indicate larval spot markings and red dotted lines outline the banding. Normal F1 larvae have a dark body with darker greyish brown banding covering wide range of the dorsal surface similar to B. mandarina. The names of spots were referred to Yoda et al. (2014). Scale bars: 1 cm.

Figure S4.
Figure S4.

Fifth instar larvae of normal F1 and B. mandarina specific apt-like mKO. Red arrows indicate ectopic white (depigmented) regions. Crescent and star spots were not depigmented in all larvae. Scale bars: 1 cm.

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Table 3.

Efficiency of allele-specific knockouts of apt-like in F1 hybrids.

Figure 5.
Figure 5.

Representative fifth instar larvae of normal F1 (left), allele-specific apt-like mKOs targeting the B. mori allele (middle) and the B. mandarina allele (right). Red arrows indicate ectopic white (depigmented) regions. Scale bars: 1 cm.

Our results have implications beyond merely confirming a role for apt-like in B. mandarina larval pigmentation. The allele-specific knockouts of apt-like in F1 hybrids allows us to compare the phenotype of genetically identical hybrids that differ only at the target locus (a framework called the “reciprocal hemizygosity test”, Steinmetz et al. 2002, Stern 2014). As such, we can attribute the loss of pigmentation in the series targeting the B. mandarina allele to evolution at the apt-like gene in B. mori, rather than exclusively at a trans-acting factor. The Apt-like proteins of B. mori (p50T) and B. mandarina (Sakado) differ by only one amino acid substitution: Alanine to Valine at residue 188 (see DDBJ accession numbers LC706749 and LC706750). However, this substitution is not observed in B. mandarina collected at different locations (see NCBI accession numbers SRR6111377, SRR6111379, SRR6111381 and SRR6111382), suggesting this is not a fixed amino acid difference between species. This implies that evolution of an apt-like cis-regulatory element contributes to the observed phenotypic difference between species.

Conclusion

Here we demonstrate the utility of CRISPR/cas9 genome editing in B. mandarina and B. mori × B. mandarina F1 hybrids to the study the function and evolution of domestication-associated candidate genes. Focusing on two pigmentation-related genes, we show that apt-like plays a role in larval body pigmentation patterning (Figure 5), whereas yellow-y plays a more general role in pigmentation that is not pattern dependent or specific to developmental life-stage (Figure 3B). These results are consistent with the proposed roles of Apt-like as a transcription factor responsible for larval color patterning, and Yellow-y as an enzyme in the melanin synthesis pathway under control of Apt-like (Futahashi et al. 2008, Yoda et al. 2014). Further, using the framework of the reciprocal hemizygosity test, we show that apt-like has evolved in B. mori in a way that has specifically reduced larval body pigmentation, without affecting the formation of larval spots or adult body pigmentation.

Despite being a powerful tool to study gene function and evolution, the allele-specific knockout approach has several limitations. First, it requires sequence variants distinguishing the parents of the F1 hybrid that result in a PAM-site that is only present in one of two species, limiting the potential to design allele-specific targets. However, this limitation may be overcome by using Cas proteins that recognize different PAM-sites (Leenay and Beisel 2017). For example, while the Streptococcus pyogenes Cas9 (SpCas9) protein has been used here, the Cas12a, which recognizes a distinct PAM sequence, has also been implemented in B. mori (Dong et al. 2020). In addition, recent studies have reported that SpCas9 can be modified to recognize alternative PAM sequences (Kleinstiver et al. 2015, 2016). A second limitation is that, given the mosacism of knockouts in G0 individuals, one cannot exclude the possibility of false-negative phenotyping results. The cleavage efficiency of the CRISPR/Cas9 system is affected by several features, such as the sequences of PAM-distal and PAM-proximal regions of the guide RNA, the genomic context of the targeted DNA, as well as GC-content and secondary structure of the guide RNA (Liu et al. 2016). To minimize this problem, one can screen a large number of G0 individuals and confirm that mutations were introduced with high efficiency (as in Figure 4 and Figure S2).

Despite these limitations, our results highlight several advantages of allele-specific knockouts in the F1 over knockouts in B. mandarina. First, our results show that B. mori (female) × B. mandarina (male) F1 embryos are substantially more tolerant to injection compared to B. mandarina. Second, allele-specific knockouts in the F1 permit the study of essential genes (such as apt-like) at which knockouts are expected to be homozygous lethal and recessive with respect to the phenotype. Finally, allele-specific CRISPR/Cas9-targeted knockouts in F1 hybrids have the added utility of identifying loci that have diverged in function between B. mori and B. mandarina, and contributing to domestication-related traits in B. mori using the framework of the reciprocal hemizygosity test. Thus, our study showcases the multifaceted utility of allele-specific knockouts in F1 hybrids in the study of gene function and evolution.

Experimental procedures

Insects

The B. mori strain p50T, a single-paired descendant of individuals of strain p50 (a derivative from Daizo; https://shigen.nig.ac.jp/silkwormbase/ViewStrainDetail.do?name=p50), is maintained at our laboratory. The B. mandarina strain, Sakado, was originally collected in Sakado-city, Saitama, Japan, in 1982. Since then, it has been maintained at our laboratory by sib-mating. All larvae were reared on fresh mulberry leaves or artificial diet (SilkMate PS, NOSAN) under continuous 12 h-light/12 h-dark conditions at 25 °C with the exceptions described below. For injections in to B. mandarina embryos, we obtained non-diapausing eggs by rearing B. mandarina larvae under continuous long-day conditions (16 h-light/8 h-dark) at 25 °C (Kobayashi 1990). We then collected eggs in crosses between emerged adults. To generate B. mori × B. mandarina F1 hybrid embryos for injection, we first incubated B. mori eggs at 15 ºC under continuous darkness (Kogure 1933). The hatched larvae were then reared under continuous 16 h-light/8 h-dark condition at 25 °C and females were crossed to B. mandarina males.

Confirmation of the yellow-y and apt-like coding sequences in B. mandarina

Total RNA was extracted from the integument of fourth instar B. mandarina larvae using TRIzol (Thermo Fisher Scientific). Complementary DNA (cDNA) was reverse transcribed from total RNA using TaKaRa RNA PCR Kit (TaKaRa). Reverse transcriptase-PCR was performed using KOD One polymerase (TOYOBO). PCR products were cloned into pGEM-T Easy Vector (Promega) and Sanger-sequenced using the FASMAC sequencing service (Kanagawa, Japan).

Knockout of yellow-y in B. mandarina

An unique crRNA target sequence in the B. mori genome was selected using CRISPRdirect (https://crispr.dbcls.jp) (Table S1) (Naito et al. 2015). The uniqueness of the target sequence in the B. mandarina genome was then confirmed by performing blastn at SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp). A mixture of crRNA, tracrRNA and Cas9 Nuclease protein NLS (600 ng/µL; NIPPON GENE) in injection buffer (100 mM KOAc, 2 mM Mg(OAc)2, 30 mM HEPES-KOH; pH 7.4) was injected into each embryo within 3 h after oviposition (Yamaguchi et al. 2011).

The injected embryos (G0 generation) were incubated at 25 °C in a humidified Petri dish until hatching. Adult G0 moths were crossed with wild-type B. mandarina, and G1 eggs were obtained. To detect heritable CRISPR/Cas9-induced mutations, ten G1 eggs were collected into one tube, and genomic DNA was prepared using the HotSHOT method (Truett et al. 2000). The region containing the target site of yellow-y crRNA was PCR-amplified using KOD One polymerase (TOYOBO). Mutations at the target site were detected by heteroduplex mobility assay using the MultiNA microchip electrophoresis system (SHIMAZU) with the DNA-500 reagent kit (Ota et al. 2013, Ansai et al. 2014).

Adult G1 moths from broods with heritable CRISPR/Cas9-induced mutations were crossed with each other to obtain homozygous knockout mutants (G2). To confirm CRISPR/Cas9-induced mutations, we prepared genomic DNA, PCR-amplified the target region and detected mutations as described above using G2 adult moths. To determine the precise nature of insertions, deletions and substitutions, PCR products obtained from G2 individuals were directly Sanger-sequenced using the FASMAC sequencing service (Kanagawa, Japan).

Comparison of post-injection hatchability

Three commonly used buffers (injection buffer 1 (Yamaguchi et al. 2011), injection buffer 2 (Tamura et al. 2000), PBS buffer) and distilled water into embryos of B. mori or B. mandarina within 3 h after oviposition. The injected embryos were incubated at 25 °C in a humidified Petri dish until hatching.

Allele-specific gene knockouts in F1 hybrids

A PAM sequence is necessary for target recognition and following DNA cleavage in CRISPR/Cas system (Hsu et al. 2013, Anders et al. 2014), and SpCas9, which we used in this study, recognizes 5”-NGG-3” as PAM. We specifically targeted SpCas9 PAM-sites that differed in sequence between B. mori and B. mandarina (Figure 4, Table S1), allowing allele-specific DNA cleavage and gene knockout (Courtney et al. 2015, Christie et al. 2017). A mixture of crRNA, tracrRNA and Cas9 was injected to F1 embryos as described above. To evaluate CRISPR/Cas9 cleavage efficiency, we extracted genomic DNA from G0 adult legs and PCR-amplified the target region as described above. PCR products were cloned into pGEM-T Easy Vector and Sanger-sequenced using an ABI3130xl genetic analyzer (Applied Biosystems).

Data Availability

Full-length coding sequences of B. mori yellow-y, B. mandarina yellow-y, B. mori apt-like and B. mandarina apt-like are available on the DDBJ under accession numbers of LC706747, LC706748, LC706749 and LC706750, respectively.

Author contributions

KT, PA and TK designed the study. KT performed most of the experiments. KT wrote the manuscript with intellectual input from TK and PA. All authors edited and approved the final version of the manuscript and agree to be accountable for all aspects of the work.

Acknowledgements

We are grateful to Katsuya Satta and Hisashi Tobita for assistance with the experiments. We are also grateful to Professor Susumu Katsuma for helpful discussions. We thank the Institute for Sustainable Agro-ecosystem Services, The University of Tokyo, for facilitating the mulberry cultivation and the Biotron Facility at the University of Tokyo for rearing the silkworms. This work was supported by JSPS KAKENHI grant number JP20J22954 to KT and JP20H02997 to TK.

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Posted May 11, 2022.
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Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval pigmentation in the domesticated silkworm, Bombyx mori
Kenta Tomihara, Peter Andolfatto, Takashi Kiuchi
bioRxiv 2022.05.07.490996; doi: https://doi.org/10.1101/2022.05.07.490996
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