Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems

• 来源期刊: 中国昆虫科学（英文版）
• 关键词: Bombyx mori CRISPR/dCas9 system transcriptional activation VP64 VPR
• 年，卷（期）: 2019,（6）
• 页码范围: 983-990
• 页数: 8页
• 语种: 英文
• DOI: 10.1111/1744-7917.12634

摘要

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms.We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR fromPrevotella andFrancisella 1 (Cpf1) could induce target mutations,deletions,inversions,and duplications both singly and multiplex in silkworm,Bombyx mori.However,it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B.mori.In this study,we investigated the CRISPRa system,in which a nuclease dead Streptococcuspyogenes Cas9 (SpCas9) is fused to two transcription activation domains,including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain),and VPR (a tripartite activator,composed of VP64,p65,and Rta).The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B.mori cells,of which the latter showed significantly higher activity.The dCas9-VPR system showed considerable activity on all five tested target genes,and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level.We also observed that this system could be used to upregulate a range of target genes.Taken together,our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B.mori and perhaps other non-drosophila insects.