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。
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Avis JM, Conn GL, Walker SC, 2012. Cis-acting ribozymes for the production of RNA in vitro transcripts with defined 5′ and 3′ ends. Methods Mol Biol, 941:83–98. https://doi.org/10.1007/978-1-62703-113-4_7
Čermák T, Curtin SJ, Gil-Humanes J, et al., 2017. A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell, 29(6):1196–1217. https://doi.org/10.1105/tpc.16.00922
Chen J, Du YN, He XY, et al., 2017. A convenient Cas9-based conditional knockout strategy for simultaneously targeting multiple genes in mouse. Sci Rep, 7:517. https://doi.org/10.1038/s41598-017-00654-2
Chen TS, Cavari B, Schartl M, et al., 2017. Identification and expression of conserved and novel RNA variants of medaka pax6b gene. J Exp Zool B (Mol Dev Evol), 328(5): 412–422. https://doi.org/10.1002/jez.b.22742
Dong FP, Xie KB, Chen YY, et al., 2017. Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells. Biochem Biophys Res Commun, 482(4):889–895. https://doi.org/10.1016/j.bbrc.2016.11.129
Fang J, Chen TS, Pan QH, et al., 2018. Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9. J Exp Zool B (Mol Dev Evol), 330(4):242–246. https://doi.org/10.1002/jez.b.22808
Feng Y, Liu S, Chen R, et al., 2021. Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(1): 73–86. https://doi.org/10.1631/jzus.B2000282
Ferreira R, Skrekas C, Nielsen J, et al., 2018. Multiplexed CRISPR/Cas9 genome editing and gene regulation using Csy4 in Saccharomyces cerevisiae. ACS Synth Biol, 7(1): 10–15. https://doi.org/10.1021/acssynbio.7b00259
Forster AC, Altman S, 1990. External guide sequences for an RNA enzyme. Science, 249(4970):783–786. https://doi.org/10.1126/science.1697102
Fujimura N, Klimova L, Antosova B, et al., 2015. Genetic interaction between Pax6 and β -catenin in the developing retinal pigment epithelium. Dev Genes Evol, 225(2): 121–128. https://doi.org/10.1007/s00427-015-0493-4
He YB, Zhang T, Yang N, et al., 2017. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing. J Genet Genomics, 44(9):469–472. https://doi.org/10.1016/j.jgg.2017.08.003
Hong YH, Liu TM, Zhao HB, et al., 2004. Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci USA, 101(21): 8011–8016. https://doi.org/10.1073/pnas.0308668101
Jao LE, Wente SR, Chen WB, 2013. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA, 110(34):13904–13909. https://doi.org/10.1073/pnas.1308335110
Jiang WY, Bikard D, Cox D, et al., 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 31(3):233–239. https://doi.org/10.1038/nbt.2508
Jinek M, Chylinski K, Fonfara I, et al., 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096):816–821. https://doi.org/10.1126/science.1225829
Knapp DJHF, Michaels YS, Jamilly M, et al., 2019. Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression. Nat Commun, 10:1490. https://doi.org/10.1038/s41467-019-09148-3
Li C, Brant E, Budak H, et al., 2021. CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(4):253–284. https://doi.org/10.1631/jzus.B2100009
Liu QZ, Yuan YM, Zhu F, et al., 2018. Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells. Biol Open, 7(8):bio035170. https://doi.org/10.1242/bio.035170
Ma J, Fan YD, Zhou Y, et al., 2018. Efficient resistance to grass carp reovirus infection in JAM-A knockout cells using CRISPR/Cas9. Fish Shellfish Immunol, 76:206–215. https://doi.org/10.1016/j.fsi.2018.02.039
Mali P, Esvelt KM, Church GM, 2013. Cas9 as a versatile tool for engineering biology. Nat Methods, 10(10):957–963. https://doi.org/10.1038/nmeth.2649
Mefferd AL, Kornepati AVR, Bogerd HP, et al., 2015. Expression of CRISPR/Cas single guide RNAs using small tRNA promoters. RNA, 21(9):1683–1689. https://doi.org/10.1261/rna.051631.115
Merenda A, Andersson-Rolf A, Mustata RC, et al., 2017. A protocol for multiple gene knockout in mouse small intestinal organoids using a CRISPR-concatemer. J Vis Exp, (125):e55916. https://doi.org/10.3791/55916
Nissim L, Perli SD, Fridkin A, et al., 2014. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell, 54(4):698–710. https://doi.org/10.1016/j.molcel.2014.04.022
Platt RJ, Chen SD, Zhou Y, et al., 2014. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 159(2):440–455. https://doi.org/10.1016/j.cell.2014.09.014
Port F, Bullock SL, 2016. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods, 13(10):852–854. https://doi.org/10.1038/nmeth.3972
Qi WW, Zhu T, Tian ZR, et al., 2016. High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol, 16:58. https://doi.org/10.1186/s12896-016-0289-2
Qin W, Liang F, Feng Y, et al., 2015. Expansion of CRISPR/Cas9 genome targeting sites in zebrafish by Csy4-based RNA processing. Cell Res, 25(9):1074–1077. https://doi.org/10.1038/cr.2015.95
Ran FA, Hsu PD, Wright J, et al., 2013. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8(11):2281–2308. https://doi.org/10.1038/nprot.2013.143
Schiffer S, Rösch S, Marchfelder A, 2002. Assigning a function to a conserved group of proteins: the tRNA 3′-processing enzymes. EMBO J, 21(11):2769–2777. https://doi.org/10.1093/emboj/21.11.2769
Shiraki T, Kawakami K, 2018. A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish. Sci Rep, 8:13366. https://doi.org/10.1038/s41598-018-31476-5
Tan JT, Zhao YC, Wang B, et al., 2020. Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining. Plant Biotechnol J, 18(11): 2161–2163. https://doi.org/10.1111/pbi.13390
Tan YY, Du H, Wu X, et al., 2020. Gene editing: an instrument for practical application of gene biology to plant breeding. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 21(6):460–473. https://doi.org/10.1631/jzus.B1900633
Tang YD, Liu JT, Wang TY, et al., 2017. CRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication. Arch Virol, 162(12):3881–3886. https://doi.org/10.1007/s00705-017-3553-4
Wang ZW, Zhu HP, Wang D, et al., 2011. A novel nucleocytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp. BMC Res Notes, 4:82. https://doi.org/10.1186/1756-0500-4-82
Wefers B, Bashir S, Rossius J, et al., 2017. Gene editing in mouse zygotes using the CRISPR/Cas9 system. Methods, 121–122:55–67. https://doi.org/10.1016/j.ymeth.2017.02.008
Xie KB, Minkenberg B, Yang YN, 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA, 112(11): 3570–3575. https://doi.org/10.1073/pnas.1420294112
Xu AT, Qin C, Lang Y, et al., 2015. A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system. Biotechnol Lett, 37(6):1265–1272. https://doi.org/10.1007/s10529-015-1796-2
Xu L, Zhao LX, Gao YD, et al., 2017. Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA. Nucleic Acids Res, 45(5):e28. https://doi.org/10.1093/nar/gkw1048
Xue T, Wang YZ, Pan QH, et al., 2018. Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus. J Fish Dis, 41(2): 365–374. https://doi.org/10.1111/jfd.12736
Yin LL, Maddison LA, Li MY, et al., 2015. Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs. Genetics, 200(2):431–441. https://doi.org/10.1534/genetics.115.176917
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).
Author information
Authors and Affiliations
Corresponding author
Additional information
Author contributions
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.
Compliance with ethics guidelines
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.
Supplementary information
Table S1; Sequences S1–S6; Fig. S1
Supplementary information
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1631/jzus.B2100343