| Summary: | The adoption of transgenic crops expressing <i>Bacillus thuringiensis</i> (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to <i>Bacillus thuringiensis</i> Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene <i>ABCC2</i> in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, <i>Ostrinia furnacalis</i>, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the <i>O. furnacalis</i> <i>ABCC2</i> (<i>OfABCC2</i>) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of <i>O. furnacalis</i> with an 8-bp deletion mutation of <i>ABCC2</i> by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of <i>OfABCC2</i> in the OfC2-KO strain. In conclusion, <i>in vivo</i> functional investigation demonstrates the causality of the <i>OfABCC2</i> truncating mutation with high-level resistance to the Cry1Fa toxin in <i>O. furnacalis</i>. Our results suggest that the <i>OfABCC2</i> protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.
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