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

SIMPLE SUMMARY: CRISPR/Cas9, a versatile gene manipulation tool, has been harnessed for targeted genome engineering in honeybees. However, until now, only SpCas9 that enables NGG recognition has been shown to manipulate the genome in A. mellifera, limiting the editable range to the NGG-included loci. In the current study, to evaluate the potential expansion when utilising Cpf1, SpCas9 and SaCas9, we predicted the distribution and number of targeting sites throughout the whole honeybee genome with a bioinformatic approach. The results of bioinformatics analysis suggest that the number of accessible targeting sites in A. mellifera could be significantly increased via the integrated CRISPR system. In addition, we measured the cleavage activity of these new CRISPR enzymes in A. mellifera, and it was found that both SaCas9 and Cpf1 can induce genome alternation in A. mellifera, albeit with relatively lower mutagenesis rates for Cpf1 and unstable editing for SaCas9. To our knowledge, our study provides the first evidence that SaCas9 and Cpf1 can efficiently mediate genome sequence mutation, thereby expanding the targetable spectrum in A. mellifera. The integrated CRISPR system will probably boost both fundamental studies and applied researches in A. mellifera and perhaps other insects. ABSTRACT: 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.