A genome sequence and efficient CRISPR/Cas9 gene editing tools for the solanaceous specialist pest Tetranychus evansi

Tetranychus evansi is an invasive spider mite pest of solanaceous crops worldwide. Its rapid global spread and ability to develop acaricide resistance highlight the need for robust genomic resources and functional genetic tools for custom control strategies. Here, we present a genome assembly and establish efficient CRISPR/Cas9 editing in T. evansi to enable mechanistic studies of host adaptation and pesticide resistance. Using an inbred line and Oxford Nanopore Technologies long-read sequencing, we assembled an 89 Mb genome into 13 contigs (N50 = 18.6 Mb) and annotated 14,246 protein-coding genes. Manual curation of detoxification gene families (P450s, CCEs, GSTs, UGTs, ABC transporters and DOGs), revealed smaller repertoires than in the polyphagous relative Tetranychus urticae. To enable reverse genetics, we adapted SYNCAS for maternal delivery of CRISPR/Cas9 in T. evansi. Targeting the phytoene desaturase (PD) pigmentation marker produced reliable knockouts with visible lack of red pigmentation and editing efficiencies of ∼10-15%, allowing the efficient creation of stable mutant lines. We further applied precision gene editing to knock-in (KI) the M918T and M918L substitutions into the voltage-gated sodium channel (VGSC). While M918L was lethal in T. evansi, we generated multiple homozygous lines for M918T (KI efficiency ∼ 4.4%). Bioassays demonstrated that while the mutation caused extremely high levels of bifenthrin resistance (RR = 345-645), this was less so for β-cyfluthrin (RR = 113-127), deltamethrin (RR = 58.2-65.6) and tau-fluvalinate (RR = 87.7-98), revealing the specific role of M918T in pyrethroid resistance. Collectively, these resources establish T. evansi as a tractable system for reverse genetic analysis and provide a reference for future comparative and population genomics.