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Journal of Integrative Agriculture  2021, Vol. 20 Issue (10): 2716-2726    DOI: 10.1016/S2095-3119(21)63736-X
Special Issue: 昆虫合辑Plant Protection—Entomolgy
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Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection
ZHANG Ting-ting1*, WEN Ting-mei1, 2*, YUE Yang1, 2, YAN Qiang3, DU Er-xia4, FAN San-hong1, Siegfried ROTH5, LI Sheng4, ZHANG Jian-zhen1, ZHANG Xue-yao1, ZHANG Min1 
1 Research Institute of Applied Biology, Shanxi University, Taiyuan 030006, P.R.China
2 School of Life Science, Shanxi University, Taiyuan 030006, P.R.China
3 State Key Laboratory of Cotton Biology/Key Laboratory of Plant Stress Biology, School of Life Sciences, Henan University, Kaifeng 475004, P.R.China
4 Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, P.R.China
5 Institute for Developmental Biology, University of Cologne, Cologne 50674, Germany
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突变效率和孵化率是影响基因编辑昆虫构建的两个关键因素。在CRISPR/Cas9介导的dsLmRNase2-/-突变体蝗虫构建过程中,我们发现注射与卵囊接触20 min的鞣化卵比未鞣化的新鲜卵获得突变体飞蝗的效率更高。然而,鞣化和未鞣化卵产生的dsLmRNase2突变遗传到G1代的效率相似。此外,发育正常的鞣化和未鞣化的G0代卵和成虫的有效突变率没有显著差异,表明鞣化并不影响CRISPR/Cas9介导的突变效率。同时,我们发现飞蝗合胞体分裂期比鞣化时间长,为显微注射的鞣化卵和未鞣化卵提供了足够的时间窗口以完成有效地基因编辑。我们进一步发现,鞣化卵显微注射后感染率较低进而表现出更高的孵化率。抗压和超微结构分析表明,鞣化卵具有压缩的卵壳,能够承受较高的外部压力。综上所述,鞣化卵具有更强的防御能力以提高孵化率,并保持了更高的基因组突变效率,为开发CRISPR/Cas9介导的飞蝗突变体构建提供了一种优化的技术方法。

The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.  In the construction of CRISPR/Cas9-mediated dsLmRNase2–/–mutant locusts, we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.  However, the heritable efficiency of the dsLmRNase2 deletion to the next generation G1 progeny was similar between adults derived from the tanned or less tanned engineered eggs.  Further, the similar effective mutant ratios in the normally developed eggs and G0 adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.  Moreover, we found that the syncytial division period, which was longer than the time for tanning, conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.  We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.  Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.  In summary, tanned eggs possess stronger defense responses and higher efficiency of genome editing, providing an improved model for developing Cas9-mediated gene editing procedures in locusts.
Keywords:  brown tanned eggs        CRISPR/Cas9        mutant locusts        microinjection        defense ability  
Received: 08 March 2021   Accepted:
Fund: This work was supported by the National Natural Science Foundation of China (32070502, 31730074, 32072419 and 31501905). 

Cite this article: 

ZHANG Ting-ting, WEN Ting-mei, YUE Yang, YAN Qiang, DU Er-xia, FAN San-hong, Siegfried ROTH, LI Sheng, ZHANG Jian-zhen, ZHANG Xue-yao, ZHANG Min. 2021. Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection. Journal of Integrative Agriculture, 20(10): 2716-2726.

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