Abstract
Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase (tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
概述
目的
探究巨细胞病毒(CMV)启动子驱动转运RNA-导向RNA(tRNA-gRNA)的表达是否能够在青鳉细胞系及胚胎中产生功能性的gRNA并诱导基因的突变。
创新点
利用CMV启动子驱动tRNA-gRNA的表达在青鳉细胞系及胚胎中产生功能性的gRNA, 结合Cas9系统诱导了青鳉胚胎和细胞系中的基因突变, 为在鱼类胚胎和细胞系中进行基因编辑提供了一种可行的方案。
方法
将tRNA-tyr gRNA(酪氨酸酶基因, tyr)或tyr gRNA与Cas9 mRNA注射到青鳉1胞期的胚胎中观察胚胎表型并通过聚合酶链式反应(PCR)和测序检测tRNA-tyr gRNA在青鳉胚胎中的功能。将pCS2-tRNA-tyr和pCS2-tRNA-PT质粒分别与Cas9 mRNA注射到青鳉1胞期的胚胎中观察胚胎表型, 并通过PCR和测序检测CMV启动子驱动的tRNA-gRNA在胚胎中的功能。将pCMV-tRNA-PT-zCas9-puro以及1:1比例的pCVpf或pCS2-tRNA-tyr与pCV-zCas9-puro的混合物分别转染SG3细胞, 通过PCR和测序检测CMV启动子驱动的tRNA-gRNA在青鳉细胞系中的功能。
结论
tRNA-tyr gRNA与tyr gRNA一样能够引起青鳉胚胎中tyr基因的突变。CMV启动子驱动tRNA-gRNA的表达在青鳉细胞系及胚胎中能够产生功能性的gRNA, 结合Cas9系统诱导青鳉胚胎和细胞系中基因的突变。
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Acknowledgments
This study was supported by the National Natural Science Foundation of China (Nos. 31771648 and 31672653), the Scientific Research Foundation of Jimei University (No. ZQ2020003), and the National Key Basic Research Program of China (No. 2013CB967700).
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Qihua PAN designed and performed the experiment, analyzed experimental data, and wrote the original draft. Junzhi LUO, Yuewen JIANG, Zhi WANG, and Ke LU participated in part of the experiment and edited the manuscript. Tiansheng CHEN designed and supervised the experiment, analyzed experimental data, wrote and edited the manuscript. All authors have read and agreed the final version of the manuscript. The authors have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Qihua PAN, Junzhi LUO, Yuewen JIANG, Zhi WANG, Ke LU, and Tiansheng CHEN declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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Table S1; Sequences S1–S6; Fig. S1
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Pan, Q., Luo, J., Jiang, Y. et al. Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA. J. Zhejiang Univ. Sci. B 23, 74–83 (2022). https://doi.org/10.1631/jzus.B2100343
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DOI: https://doi.org/10.1631/jzus.B2100343