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Journal of Integrative Agriculture  2024, Vol. 23 Issue (6): 2006-2017    DOI: 10.1016/j.jia.2023.06.039
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CRISPR/Cas9-mediated NlInR2 mutants: Analyses of residual mRNA and truncated proteins

Jun Lü1, 2, Jingxiang Chen1, Yutao Hu1 , Lin Chen1, Shihui Li1, Yibing Zhang1, Wenqing Zhang1#

State Key Laboratory of Biocontrol/School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China

Development of Food Science and Engineering, Moutai Institute, Renhuai 564507, China

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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.
Keywords:  CRISPR/Cas9        Nilaparvata lugens        residual mRNA        skipping exon        truncated protein   
Received: 12 April 2023   Accepted: 29 May 2023
Fund: We gratefully thank the National Natural Science Foundation of China (31730073).
About author:  Jun Lü, E-mail: lyuj123@yeah.net; #Correspondence Wenqing Zhang, E-mail: lsszwq@mail.sysu.edu.cn

Cite this article: 

Jun Lü, Jingxiang Chen, Yutao Hu , Lin Chen, Shihui Li, Yibing Zhang, Wenqing Zhang. 2024.

CRISPR/Cas9-mediated NlInR2 mutants: Analyses of residual mRNA and truncated proteins . Journal of Integrative Agriculture, 23(6): 2006-2017.

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