Transcript Level and Sequence Matching Are Key Determinants of Off-Target Effects in RNAi

Jiasheng Chen, Cheng Wang Sheng, Yingchuan Peng, Kangxu Wang, Yaoyu Jiao, Subba Reddy Palli, Haiqun Cao

Research output: Contribution to journalArticlepeer-review


Double-stranded RNA (dsRNA) pesticides, those based on RNA interference (RNAi) technology utilizing dsRNA, have shown potential for pest control. However, the off-target effects of dsRNA pose limitations to the widespread application of RNAi and raise concerns regarding potential side effects on other beneficial organisms. The precise impact and underlying factors of these off-target effects are still not well understood. Here, we found that the transcript level and sequence matching jointly regulate off-target effects of dsRNA. The much lower expressed target genes were knocked down to a lesser extent than genes with higher expression levels, and the critical sequence identity of off-target effects is approximately 80%. Moreover, off-target effects could be triggered by a contiguous matching sequence length exceeding 15 nt as well as nearly perfectly matching sequences with one or two base mismatches exceeding 19 nt. Increasing the dosage of dsRNA leads to more severe off-target effects. However, the length of mismatched dsRNA, the choice of different RNAi targets, and the location of target sites within the same gene do not affect the severity of off-target effects. These parameters can be used to guide the design of possibly selective sequences for RNAi, optimize the specificity and efficiency of dsRNA, and facilitate practical applications of RNAi for pest control.

Original languageEnglish
Pages (from-to)577-589
Number of pages13
JournalJournal of Agricultural and Food Chemistry
Issue number1
StatePublished - Jan 10 2024

Bibliographical note

Publisher Copyright:
© 2023 American Chemical Society.


  • RNA interference
  • Tribolium castaneum
  • chimeric dsRNA
  • dsRNA pesticides
  • sequence identity

ASJC Scopus subject areas

  • General Chemistry
  • General Agricultural and Biological Sciences


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