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Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) <jats:italic>\u03b16<\/jats:italic> subunit strongly associated with spinosyns resistance in insects, including <jats:italic>Plutella xylostella<\/jats:italic>. However, reverse genetic verification of the <jats:italic>P. xylostella nAChR\u03b16<\/jats:italic> has not yet been achieved via an <jats:italic>in vivo<\/jats:italic> approach.<\/jats:p><\/jats:sec><jats:sec><jats:title>RESULTS<\/jats:title><jats:p>Here, we successfully constructed a homozygous strain (Px\u03b16\u2010KO) with a 2\u2010nt deletion mutation of <jats:italic>nAChR\u03b16<\/jats:italic> by CRISPR\/Cas9 coupled with non\u2010homologous end joining approach in <jats:italic>P. xylostella<\/jats:italic>. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3\u2013TM4 loop region. When compared to the background strain IPP\u2010S, the knockout strain Px\u03b16\u2010KO exhibited 229\u2010 and 1462\u2010fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios &lt;3\u2010fold) effects on the toxicities of imidacloprid, abamectin, \u03b2\u2010cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2\u2010nt deletion mutation of <jats:italic>nAChR\u03b16<\/jats:italic> in the Px\u03b16\u2010KO strain.<\/jats:p><\/jats:sec><jats:sec><jats:title>CONCLUSION<\/jats:title><jats:p><jats:italic>In vivo<\/jats:italic> functional investigation demonstrates the causality of the <jats:italic>Px\u03b16<\/jats:italic> truncating mutation with high levels of resistance to spinosyns in <jats:italic>P. xylostella<\/jats:italic>. Our results suggest the Px\u03b16\u2010KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. \u00a9 2019 Society of Chemical Industry<\/jats:p><\/jats:sec>","DOI":"10.1002\/ps.5689","type":"journal-article","created":{"date-parts":[[2019,11,22]],"date-time":"2019-11-22T12:59:51Z","timestamp":1574427591000},"page":"1618-1625","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":56,"title":["Disruption of nicotinic acetylcholine receptor \u03b16 mediated by CRISPR\/Cas9 confers resistance to spinosyns in <i>Plutella xylostella<\/i>"],"prefix":"10.1002","volume":"76","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4775-0100","authenticated-orcid":false,"given":"Xingliang","family":"Wang","sequence":"first","affiliation":[{"name":"College of Plant Protection Nanjing Agricultural University  Nanjing 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