Expansion of CRISPR Targeting Sites Using an Integrated Gene-Editing System in Apis mellifera.

CRISPR/Cas9, a predominant gene-editing tool, has been utilised to dissect the gene function in Apis mellifera. However, only the genomic region containing NGG PAM could be recognised and edited in A. mellifera, seriously hampering the application of CRISPR technology in honeybees. In this study, we carried out the bioinformatics analysis for genome-wide targeting sites of NGG, TTN, and NNGRRT to determine the potential expansion of the SpCas9, SaCas9, Cpf1, and it was found that the targetable spectrum of the CRISPR editing system could be markedly extended via the integrated gene manipulation system. Meanwhile, the single guide RNA (sgRNA)/crRNA of different novel gene editing systems and the corresponding CRISPR proteins were co-injected into honeybee embryos, and their feasibility was tested in A. mellifera. The sequencing data revealed that both SaCas9 and Cpf1 are capable of mediating mutation in A. mellifera, albeit with relatively lower mutagenesis rates for Cpf1 and unstable editing for SaCas9. To our knowledge, our results provide the first demonstration that SaCas9 and Cpf1 can function to induce genome sequence alternation, which extended the editing scope to the targets with TTN and NNGRRT and enabled CRISPR-based genome research in a broader range in A. mellifera.

Transcriptional Profiles of Diploid Mutant Apis mellifera Embryos after Knockout of csd by CRISPR/Cas9.

In honey bees, complementary sex determiner (csd) is the primary signal of sex determination. Its allelic composition is heterozygous in females, and hemizygous or homozygous in males. To explore the transcriptome differences after sex differentiation between males and females, with genetic differences excluded, csd in fertilized embryos was knocked out by CRISPR/Cas9. The diploid mutant males at 24 h, 48 h, 72 h, and 96 h after egg laying (AEL) and the mock-treated females derived from the same fertilized queen were investigated through RNA-seq. Mutations were detected in the target sequence in diploid mutants. The diploid mutant drones had typical male morphological characteristics and gonads. Transcriptome analysis showed that several female-biased genes, such as worker-enriched antennal (Wat), vitellogenin (Vg), and some venom-related genes, were down-regulated in the diploid mutant males. In contrast, some male-biased genes, such as takeout and apolipophorin-III-like protein (A4), had higher expressions in the diploid mutant males. Weighted gene co-expression network analysis (WGCNA) indicated that there might be interactions between csd and fruitless (fru), feminizer (fem) and hexamerin 70c (hex70c), transformer-2 (tra2) and troponin T (TpnT). The information provided by this study will benefit further research on the sex dimorphism and development of honey bees and other insects in Hymenoptera.

CRISPR/Cas9 mediated knockout of Amyellow-y gene results in melanization defect of the cuticle in adult Apis mellifera.

Visible genetic markers are critical to gene function studies using genome editing technology in insects. However, there is no report about visible phenotypic markers in Apis mellifera, which extremely influences the application of genomic editing in honey bees. Here, we cloned and characterized the Amyellow-y gene in A. mellifera. Stage expression profiles showed that Amyellow-y gene was highly expressed in 2-, 4-day-old pupae, and newly emerged bees, and a high expression level was detected in the leg, thorax, wing and sting. To understand its functional role in pigmentation, Amyellow-y edited honeybees were created using CRISPR/Cas9, and it was found that the black pigment was decreased in the cuticle of mosaic workers and mutant drones. In particular, mutant drones manifested an overall appearance of yellowish cuticle in the body and appendages, including antennae, wings and legs, indicating that mutagenesis induced by disruption of Amyellow-y with CRISPR/Cas9 are heritable. Furthermore, the expression levels of genes associated with melanin pigmentation was investigated in mutant and wild-type drones using quantitative reverse transcription PCR. Transcription levels of Amyellow-y and aaNAT decreased markedly in mutant drones than that in wild-type ones, whereas laccase 2 was significantly up-regulated. Our results provide the first evidence, to our knowledge, that CRISPR/Cas9 edited G1 mutant drones of A. mellifera have a dramatic body pigmentation defect that can be visualized in adults, suggesting that Amyellow-y may serve as a promising visible phenotypic marker for genome editing in honey bees.