CRISPR/Cas9-mediated NlInR2 mutants: Analyses of residual mRNA and truncated proteins

Abstract

CRISPR/Cas9 technology is a powerful genome manipulation tool in insects. However, little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants. This study generated homozygous mutants of the insulin receptor gene 2 (NlInR2) in the brown planthopper (Nilaparvata lugens) using CRISPR/Cas9 genome editing. Both frameshift mutants, E5_D17 and E6_I7, differentiated towards long wings, but there were differences in wing morphology, with E5_D17 showing wing deformities. Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants, as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17. Additionally, the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein. RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%, suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions. Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.