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 homozy...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.展开更多
基金the National Natural Science Foundation of China(31730073).
文摘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.
