CRISPR/Cas9‐mediated saturated mutagenesis of the cotton MIR482 family for dissecting the functionality of individual members in disease response

Many genes encoding nucleotide‐binding leucine‐rich repeat receptors (NLRs) are regulated and fine‐tuned by miR482 to balance the trade‐off between disease resistance and growth. Dicotyledonous plants, including cotton, usually have multiple miR482 isoforms. Each miR482 isoform can regulate several NLRs that in turn can be regulated by several different miR482 isoforms. Dissecting the functionality of individual miR482 isoforms in disease response and in balancing the disease resistance and growth trade‐off demands a collection of mutants mutated in individual miR482 members (single or multiple). In this study, we generated such a collection of cotton miR482 mutants using CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) genome editing and transformation of pooled guide RNAs (gRNAs). In total, 84 T(0) plants representing 40 independent transgenic events and harboring mutation in each of the 10 miR482 isoforms were generated. The average editing efficiency of the 18 transformed gRNAs is 75%, ranging from 0 (3 gRNAs) to 100% (8 gRNAs). Most miR482 isoforms have a diverse range of mutations, including small indels (1–44 bp) and substitutions, which are expected to impair biogenesis of miR482. All nine mutant populations used in Verticillium dahliae infection experiments showed a disease index lower than the control, with four being significantly lower. The disease assay also suggests a different role of different miR482 isoforms in disease response and a potential dosage effect of miR482l. The study demonstrates the feasibility of saturation mutagenesis of plant miRNA families with dozens of genetic loci using CRISPR/Cas9 and provides the cotton community a valuable resource for uncovering the miR482‐NLR module(s) underlying the interaction between cotton and different pathogens.