Objective To construct miR-223 full-sequence-knockout mice with dual sgRNAs. Methods DualsgRNAs were designed and transcribed in vitro, then microinjected into zygotes of C57BL/6 mice with Cas9 mRNA. After themice were born, genomic DNA was subjected to PCR and sequenced to identify their genotypes. In addition, total RNA wasextracted from the livers of mice, and expression of miR-223 in the liver tissues was analyzed by real-time PCR. Results Dual miR-223 sgRNAs were designed and transcribed in vitro. After purification, mouse zygotes were microinjected to obtainmiR-223 mutant mice. Sequencing result revealed that the mutant mice had three genotypes: one with a 6-bp deletion that didnot affect the miR-223 sequence, and two with 162-bp and 168-bp deletions, respectively, resultsing in full-sequence-deletionof miR-223. Compared with wild-type mice, miR-223 expression was barely detected in liver tissues of the two full-deletion mice. Conclusions miR-223 full-sequence-knockout mice are successfully generated using dual sgRNAs.