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Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species.Genetic engineering in pigs holds a great promise in biomedical research.By co-injection of one-cell stage embryos with Cas9 mRNA and Npc111 sgRNA,we achieved precise Npc111 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%.Meanwhile,we carefully analyzed theNpc111 gRNA:Cas9-mediated on-and off-targetmutations in various somatic tissues and ovaries,and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.