CRISPR/Cas9-mediated NlInR2 mutants: Analysesof residual mRNA and truncated proteins
2024
Jun Lü | Jingxiang Chen | Yutao Hu | Lin Chen | Shihui Li | Yibing Zhang | Wenqing Zhang
CRISPR/Cas9 technologyis a powerful genome manipulation tool in insects. However, little is known about whether mRNAand protein of a target gene are completely cleared in homozygous mutants. This study generated homozygous mutants ofthe insulin receptor gene 2 (NlInR2) in the brown planthopper (Nilaparvata lugens)using CRISPR/Cas9 genome editing. Bothframeshift mutants, E5_D17 and E6_I7, differentiated towards long wings, butthere were differences in wing morphology, with E5_D17 showing wingdeformities. Subsequent investigationsrevealed the presence of residual expression of NlInR2 mRNA in bothmutants, as well as the occurrence of spliceosomes featuring exon skippingsplicing in E5_D17. Additionally, theE5_D17 exhibited the detection of N-terminally truncated NlInR2 protein. RNA interference experiments indicated thatthe knockdown of NlInR2 expression in the E5_D17 mutant line increasedthe proportion of wing deformities from 11.1 to 65.6%, suggesting that theresidual NlInR2 mRNA of the E5_D17 mutant might have retained somegenetic functions. Our results implythat systematic characterization of residual protein expression or function inCRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.
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