CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab

Abstract

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Figure 1

Mutations in 28 PgABCA2 cDNA sequences from five Cry2Ab-resistant pink bollworm larvae from the CRISPR-R2 strain that lacked gDNA mutations in sgRNA target sites. (A) The predicted PgABCA2 protein includes amino (N) and carboxyl (C) termini and transmembrane domains (TMD1 and TMD2). Each TMD contains six transmembrane-spanning regions (TM, green), three extracellular loops (ECL, purple), and two intracellular loops (ICL, blue). The two TMDs are connected by a single intracellular loop (ICL3). ICL3 and the C-terminal domain each contain a nucleotide-binding domain (NBD, orange). (B) Mutations in PgABCA2 cDNAs from CRISPR-R2 (3–8 clones from each of five individuals: 3, 5, 9, 10 and 18) relative to the susceptible strain APHIS-S (MG637361). Individuals with two distinct PCR products cloned are indicated as A or B (e.g., 5A and 5B, etc.). Numbers to the right of the decimal point for each individual indicate the clone sequenced. Exon numbers are shown for APHIS-S. Location of sgRNAs 1–5 are shown as colored bars (sgRNA1, magenta; sgRNA2, cyan; sgRNA3, blue; sgRNA4, yellow; sgRNA5, red). Red triangles above bars indicate premature stop codons, which occur in all sequences except 10.1, 10.21, 10.22, and 10.24. Colors within bars show mutations: orange for insertions and deletions (indels), red for deletions, and green for insertions.

Figure 2

Mutations in 12 PgABCA2 cDNA sequences from six Cry2Ab-resistant pink bollworm larvae from F₂ progeny of CRISPR-R2 X APHIS-S single-pair families. Mutations in PgABCA2 cDNAs from CRISPR-R2 survivors from three single-pair families (1–3 clones from each of five individuals from families A, C, and J) relative to the susceptible strain APHIS-S (MG637361). Numbers to the right of the family name indicates individual and clone number, respectively (e.g., A.1.1 indicates Family A, Individual 1, Clone 1). Exon numbers are shown for APHIS-S. Location of sgRNAs 1–5 are shown as colored bars (sgRNA1, magenta; sgRNA2, cyan; sgRNA3, blue; sgRNA4, yellow; sgRNA5, red). Red triangles above bars indicate premature stop codons. Colors within bars show mutations: orange for insertions and deletions (indels), red for deletions, and green for insertions.