利用化脓链球菌成簇的规则间隔短回文重复序列相关蛋白9（spCas9）/转运RNA（tRNA）-导向RNA（gRNA）（SpCas9/tRNA-gRNA）在青鳉（Oryzias latipes）细胞系和胚胎中进行高效基因编辑

Efficient gene editing in a medaka ( Oryzias latipes ) cell line and embryos by SpCas9/tRNA-gRNA 利用化脓链球菌成簇的规则间隔短回文重复序列相关蛋白9（spCas9）/转运RNA（tRNA）-导向RNA（gRNA）（SpCas9/tRNA-gRNA）在青鳉（ Oryzias latipes ）细胞系和胚胎中进行高效基因编辑 1 2 first-author 2 2 2 2 1 2 corresp Tiansheng CHEN, tiansheng.chen@jmu.edu.cn Tiansheng CHEN, tiansheng.chen@jmu.edu.cn tiansheng.chen@jmu.edu.cn https://orcid.org/0000-0003-4763-2307 Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Fisheries College, Jimei University, Xiamen361021, China Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Fisheries College, Jimei University, Xiamen361021, China College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan430070, China College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan430070, China 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. 目的： 2 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： 2 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： 2 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： 2 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。 Medaka ( Oryzias latipes ) Gene editing Poly-tRNA-gRNA Embryos Fish cells 青鳉（ Oryzias latipes ） 基因编辑 Poly-tRNA-gRNA 胚胎 鱼类细胞系 1 Introduction 1 Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) form a CRISPR/Cas system, which is an important acquired immune system for bacteria ( Jinek et al., 2012 Jinek et al., 2012 ; Feng et al., 2021 Feng et al., 2021 ). One type of CRISPR/Cas system, the modified CRISPR/Cas9 system, has evolved into a powerful genome-editing tool ( Li et al., 2021 Li et al., 2021 ). Cas9 protein directly cleaves the target DNA sequence under the guidance of a guide RNA (gRNA), enabling nonhomologous end joining (NHEJ), homology-directed repair (HDR) ( Mali et al., 2013 Mali et al., 2013 ; Ran et al., 2013 Ran et al., 2013 ), or microhomology-mediated end joining (MMEJ) ( Tan JT et al., 2020 Tan JT et al., 2020 ). This results in insertion and deletion at the target site in the genomes of eukaryotes ( Platt et al., 2014 Platt et al., 2014 ; Wefers et al., 2017 Wefers et al., 2017 ), prokaryotes ( Jiang et al., 2013 Jiang et al., 2013 ), and viruses ( Xu et al., 2015 Xu et al., 2015 ; Tang et al., 2017 Tang et al., 2017 ). To achieve gene knockout in particular tissues or simultaneous multiple gene knockout with the CRISPR/Cas9 system, researchers have used the specific promoter to control expression of the Cas9 gene or used multiple RNA polymerase Ⅲ promoters (such as U6 promoter, 7SK promoter, and H1 promoter) to control the expression of different gRNAs ( Chen J et al., 2017 Chen J et al., 2017 ; Merenda et al., 2017 Merenda et al., 2017 ). However, the function and sequence of these promoters are not conserved across different species according to previous research ( Liu et al., 2018 Liu et al., 2018 ). Recently, some strategies were tried to generate multiple functional gRNAs using some elements. Endonuclease CRISPR system Yersinia ribonuclease 4 (Csy4) protein was used to efficiently synthesize gRNA in yeast ( Ferreira et al., 2018 Ferreira et al., 2018 ), plants (Čermák et al., 2017), zebrafish ( Qin et al., 2015 Qin et al., 2015 ), and human cells ( Nissim et al., 2014 Nissim et al., 2014 ). However, in zebrafish, all injected embryos exhibited severe deformity after co-injection of Csy4 messenger RNA (mRNA), Cas9 mRNA, and Csy gRNA ( Qin et al., 2015 Qin et al., 2015 ). Ribozymes can also enable efficient generation of gRNA and induce conditional gene knockout under the control of a specific RNA polymerase II-dependent promoter ( He et al., 2017 He et al., 2017 ). Hammerhead (HH) ribozyme and hepatitis delta virus (HDV) ribozyme were simultaneously used to cleave RNA due to their respective 5'-end and 3'-end digestion activity ( Avis et al., 2012 Avis et al., 2012 ). The transfer RNA (tRNA)-linked transcript was also cleaved at the 5'-end and 3'-end of tRNA by RNaseP and RNaseZ, respectively ( Forster and Altman, 1990 Forster and Altman, 1990 ; Schiffer et al., 2002 Schiffer et al., 2002 ), which made the tRNA-linked gRNA shorter than the HH+HDV-linked gRNA. Thus, the tRNA-mediated CRISPR/Cas9 system has been used to generate multiple gene knockout lines in plants ( Xie et al., 2015 Xie et al., 2015 ; Qi et al., 2016 Qi et al., 2016 ), Drosophila ( Port and Bullock, 2016 Port and Bullock, 2016 ), zebrafish ( Shiraki and Kawakami, 2018 Shiraki and Kawakami, 2018 ), and human cells ( Knapp et al., 2019 Knapp et al., 2019 ). Moreover, under the control of RNA polymerase II promoter, the tRNA-gRNA transcript achieved multiple gene knockout in specific mouse tissues ( Xu et al., 2017 Xu et al., 2017 ). Undoubtedly, the tRNA-gRNA system is an ideal solution for multiple or conditional gene knockout. Recently, the CRISPR/Cas9 system was used to generate a knockout fish line via injecting a gRNA and a modified Cas9 mRNA into one-cell stage medaka ( Oryzias latipes ) embryos ( Fang et al., 2018 Fang et al., 2018 ). However, gene editing methods for multiple genes or cell lines have rarely been reported in medaka. Here, we used poly-tRNA-gRNA (PTG) combined with the CRISPR/Cas9 system to induce gene editing in medaka embryos and in a fish cell line, spermatogonial cell line (SG3). Our results provid a tool for achieving gene editing in cell lines or multiple gene knockout in medaka. 2 Materials and methods 1 2.1 Fish and embryos 2 The medaka fish HdrR line was raised at Laboratory of Jimei University (Xiamen, China) and maintained in a 28 ℃ water circulation system with a photoperiod of 14-h light/10-h darkness ( Chen TS et al., 2017 Chen TS et al., 2017 ). The embryos were collected from the adult fish and cultured in embryonic rearing medium until the larvae were hatched. All procedures complied with the protocol approved by the Animal Care and Use Committee of Jimei University (Xiamen, China). 2.2 Vector construction 2 Based on the previous study ( Xie et al., 2015 Xie et al., 2015 ), we used a string of rice tRNA sequence to generate the expression vectors for this study. The pCS2-tRNA- tyr plasmid was constructed as follows: a string of the sequence tRNA: Bbs I- Bbs I:scaffold-tRNA: tyr gRNA:scaffold (Sequences S1‍–‍S4; Fang et al., 2018 Fang et al., 2018 ) was synthesized in GenScript ( https://www.genscript.com.cn https://www.genscript.com.cn ). We then inserted the string into pCS2+ plasmid digested by Bam HI and Xho I, and sequenced it ( Fig. 1 Fig. 1 a). A DNA fragment containing medaka pax6.1 gRNA scaffold was amplified with polymerase chain reaction (PCR) (primer: PT-F/PT-R; Table S1) and cloned into the Bbs I site of the pCS2-tRNA- tyr (GenBank accession number: MW827168) to construct pCS2-tRNA- pax6.1 gRNA (Sequence S5)-tRNA- tyr gRNA (pCS2-tRNA-PT; GenBank accession number: MW827169) ( Fig. 2 Fig. 2 a). To construct the pCV-zCas9 plasmid, we amplified the fragment of cytomegalovirus (CMV)‍-T7 (CMV-T7 F/CMV-T7 R; Table S1) and inserted it upstream of zebrafish codon-optimized Cas9 (zCas9) in pT3TS-nls-zCas9-nls ( Jao et al., 2013 Jao et al., 2013 ; Fang et al., 2018 Fang et al., 2018 ). We amplified the sequence of 2A-puromycin from pX462 (Addgene: #48141) (zCas9 Afe I F/zCas9 Afe I R, zCas9 puro F/zCas9 puro R; Table S1) and inserted it downstream of zCas9 in pCV-zCas9, which formed pCV-zCas9-puro (GenBank accession number: MW827170) ( Ran et al., 2013 Ran et al., 2013 ; Fang et al., 2018 Fang et al., 2018 ). The vector pCMV-tRNA-PT-zCas9-puro (GenBank accession number: MW827171) was constructed by inserting a CMV-tRNA- pax6.1 gRNA-scaffold-tRNA- tyr gRNA-scaffold (Sequence S6) fragment into the CMV element of pCV-zCas9-puro plasmid (primer: all-F/all-R; Table S1, Fig. 3 Fig. 3 a). We designed the pax6.1 gRNA using the CHOPCHOP website ( https://chopchop.cbu.uib.no https://chopchop.cbu.uib.no ). The pCVpf vectors were generously given by Prof. Yunhan HONG, National University of Singapore. 2.3 Cell culture and transfection 2 The SG3 cell line generously given by Prof. Yunhan HONG was maintained in our lab. The cells were cultured at 28 ℃ in germ cell culture medium, which consisted of Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose and supplemented with 20 mmol/L hydroxyethyl piperazineethanesulfonic acid (HEPES; pH 7.4), glutamine (2 mmol/L), Na-pyruvate (1 mmol/L), Na-selenite (2 nmol/L), non-essential amino acids (1 mmol/L), 2-mercaptoethanol (100 μmol/L), human recombinant β fibroblast growth factor (10 ng/mL), antibiotics (penicillin, 100 U/mL; streptomycin, 100 μg/mL), fish embryos extracted from medaka, 15% (volume fraction) fetal bovine serum, and 1% (volume fraction) fish serum from trout ( Hong et al., 2004 Hong et al., 2004 ). The cells were seeded at 7×10 5 cell/mL into six-well plates (70% confluence) before transfection. Then, cells were transfected with 2 µg of plasmid DNA (pCVpf or pCS2-tRNA- tyr :pCV-zCas9-puro at 1:1 (mass ratio); pCMV-tRNA-PT-zCas9-puro) using Lipofectamine TM 2000 transfection reagent (11668019; Invitrogen, USA) ( Xue et al., 2018 Xue et al., 2018 ). After two days of transfection, the cells were selected with puromycin at 2 μg/mL for two weeks. The cells were observed using Ti-U inverted fluorescent microscopy (Nikon, Tokyo, Japan). 2.4 Microinjection 2 The tyr gRNA template was amplified using tyr gRNA F and tyr gRNA R (Table S1), and the tyr gRNA was prepared from the template with a TranscriptAid T7 High Yield Transcription kit (K0441; Thermo Scientific, USA). The pCS2-tRNA- tyr plasmid was digested by Xho I to form a template for in vitro transcription of tRNA- tyr gRNA. To prepare tRNA- tyr gRNA, we used a mMESSAGE mMACHINE TM SP6 Transcription kit (AM1340; Invitrogen). The Cas9 mRNA was produced from the pZCas9 ( Fang et al., 2018 Fang et al., 2018 ) using a mMESSAGE mMACHINE TM T7 Transcription kit (AM1344; Invitrogen). The mixture containing 100 ng/µL gRNA or 30 ng/µL plasmid, 500 ng/µL Cas9 mRNA, and 500 µg/mL phenol red was injected into one-cell stage embryos. The injected embryos were then cultured in embryonic rearing medium at 28 ℃ in an incubator, and the embryos were collected 5 d post-injection (dpi) to extract genomic DNA. We imaged the embryos with a Leica M205 FA stereomicroscope (Leica, Germany). 2.5 Mutation screening 2 The genomic DNAs of the cells and embryos were extracted as previously described ( Wang et al., 2011 Wang et al., 2011 ). About 5 dpi embryos or cells from six wells were used to extract the genomic DNA. Each sample was lysed with cell lysis buffer (10 mmol/L Tris-HCl (pH 8.0), 75 mmol/L NaCl, 5 mmol/L ethylenediamine tetraacetic acid (EDTA), and 5 mg/mL sodium dodecyl sulfate (SDS)) and proteinase K (100 µg/mL) at 65 ℃ for 3 h. Proteins and other impurities were removed by adding NH 4 Ac (7.5 mol/L), and then the DNA was precipitated with isopropanol and resuspended in TE (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 8.0). The target fragment was amplified with PCR ( Fang et al., 2018 Fang et al., 2018 ). The primers are listed in Table S1. The PCR program was set at 95 ℃ for 3 min, followed by 35 cycles of 95 ℃ for 30 s, 60 ℃ for 30 s, and 72 ℃ for 30 s, then 72 ℃ for 10 min. The PCR products were digested by T7 Endonuclease I (EN303-01; Vazyme, China) to detect mutation and then cloned into pMD18-T (D103A; TaKaRa, Japan) for single-colony sequencing. The sequences were analyzed with the SnapGene program ( https://www.snapgene.com https://www.snapgene.com ). 3 Results 1 3.1 Activity of tRNA-gRNA RNA in medaka embryos 2 To explore whether CRISPR/Cas9 combined with the tRNA-based RNA procession system can cause gene editing in medaka, we synthesized a fragment including rice tRNA ligated by the tyr gRNA of medaka and inserted it into the pCS2+ plasmid to construct the pCS2-tRNA- tyr vector. We produced tRNA- tyr gRNA, Cas9 mRNA, and tyr gRNA by in vitro transcription from the pCS2-tRNA- tyr vector, pZCas9 plasmid, and tyr gRNA template, respectively ( Fig. 1 Fig. 1 a). Then, we co-injected tRNA- tyr gRNA and Cas9 mRNA into the one-cell stage embryos, which showed mosaic loss of retinal pigment in the tRNA- tyr gRNA group. The injected tyr gRNA group, as the positive control, also exhibited mosaic loss of retinal pigment (Figs. 1b 1b and 1c 1c ). In contrast, the normal pigment existed in the uninjected embryo group (Figs. 1b 1b and 1c 1c ). To further verify that the loss of pigment was due to mutation of the tyr gene, we amplified the fragments containing the tyr gRNA target site and subjected them to Sanger sequencing. The result showed that the mutation sequence was indeed located at the gRNA target site ( Fig. 1 Fig. 1 d), and produced at least three different types of mutants (-7 bp, -5 bp, and -5/+7 bp). Statistical data for pigment loss placed about 74% ( n =32/43) of individuals in the injected tRNA- tyr gRNA group. By contrast, the injected tyr gRNA group appeared to exhibit higher mutation efficiency, with a mutation rate of 100% ( n =22/22) ( Fig. 1 Fig. 1 b). These results suggested that the transcribed tRNA-gRNA, similar to the gRNA, induced DNA editing in medaka embryos. 3.2 tRNA-mediated multiple gene editing in medaka embryos 2 To investigate the feasibility of expressing multiple functional gRNAs via CMV promoter, we constructed the pCS2-tRNA-PT vector by inserting pax6.1 gRNA in the Bbs I site of pCS2-tRNA- tyr ( Fig. 2 Fig. 2 a). In our previous study (unpublished data), pax6.1 gRNA was validated as producing the aniridic eye in medaka, which is a canonical and visible phenotype for observation during eye development (Fig. S1). We co-injected Cas9 mRNA with either plasmid pCS2-tRNA- tyr (group A) or pCS2-tRNA-PT (group B) into the one-cell stage embryos. In the group A (pCS2-tRNA- tyr and zCas9 mRNA), we noted deletion of pigment ( Fig. 2 Fig. 2 c). In the group B (pCS2-tRNA-PT and zCas9 mRNA), we also found deletion of pigment and abnormal development of the eye ( Fig. 2 Fig. 2 c). Subsequently, amplification of the target site and monoclonal sequencing confirmed that these phenotypes resulted from mutation of tyr and pax6.1 (Figs. 2d 2d and 2e 2e ). Additionally, statistical data on the pigment deletion showed that the mutation ratio of the injected pCS2-tRNA- tyr and zCas9 mRNA group approximated 46% ( n =47/102), while the injected pCS2-tRNA-PT and zCas9 mRNA group showed higher mutation efficiency, with a mutation rate of 75% ( n =45/60) ( Fig. 2 Fig. 2 b). The latter result was caused by the knockout of pax6.1 , which also led to blocked pigmentation ( Fujimura et al., 2015 Fujimura et al., 2015 ). The mutant embryos were grown to adulthood and then intercrossed to generate albino fish ( Fig. 2 Fig. 2 f). The albino fish had red eyes while the wild type still had black eyes. Thus, the tRNA-mediated gene editing enabled us to obtain fertile mutation generations. These results indicated that the tRNA-gRNA system could also produce functional gRNA under the control of CMV promoter in medaka embryos, either by RNA injection or DNA injection. Moreover, the vector pCS2-tRNA- tyr ( Bbs I) provided one-step cloning to knock out any candidate genes. 3.3 tRNA-mediated gene editing in the fish cell line 2 Many strategies for Cas9-mediated gene editing in cells involve transfecting the vector containing U6 promoter to express gRNA stably ( Yin et al., 2015 Yin et al., 2015 ). However, the U6 promoter is not conservative across the phyla ( Liu et al., 2018 Liu et al., 2018 ), whereas the above results indicated that the tRNA-gRNA system can also produce functional gRNA under the control of CMV promoter. Thus, we tested the feasibility of this strategy in the fish cell line medaka SG3. We explored three different combinations of plasmids: (1) the all-in-one plasmid pCMV-tRNA-PT-zCas9-puro ( Fig. 3 Fig. 3 a), (2) two separated plasmids pCV-zCas9-puro and pCS2-tRNA- tyr , and (3) pCVpf and pCV-zCas9-puro, as control. We transfected each of these plasmid combinations into SG3 cells ( Fig. 3 Fig. 3 b). Through puromycin selection, we extracted the DNA from transfected cells to amplify the target region of tyr . After amplification, the products could be digested by T7 endonuclease I ( Fig. 3 Fig. 3 c), which suggested that the pCMV-tRNA-PT-zCas9-puro and the mixture of pCV-zCas9-puro and pCS2-tRNA- tyr induced mutation of the tyr target site. In the subsequent sequencing, the tyr mutation sequence was detected. In the pCMV-tRNA-PT-zCas9-puro group, we found the +2 bp mutation type, and at least four types of mutation were also present in the pCV-zCas9-puro and pCS2-tRNA- tyr group (-6 bp, -20/+25 bp, -10 bp, +4 bp) ( Fig. 3 Fig. 3 d). Therefore, the CMV promoter can also drive transcription of the tRNA-gRNA system in medaka SG3 cells, and produce functional gRNA to edit the target gene. 4 Discussion 1 Gene editing induced by the CRISPR/Cas9 system has been found in many organisms ( Jiang et al., 2013 Jiang et al., 2013 ; Xu et al., 2015 Xu et al., 2015 ; Fang et al., 2018 Fang et al., 2018 ; Tan YY et al., 2020 Tan YY et al., 2020 ). Recently, the PTG system has been employed to widen the field of application of the Cas9 system. In rice, the PTG system under the control of RNA polymerase III promoter was used to induce the mutation of multiple genes ( Xie et al., 2015 Xie et al., 2015 ). Subsequently, the rice tRNA sequence was directly used to generate the mutant of genomic DNA in Drosophila ( Port and Bullock, 2016 Port and Bullock, 2016 ), zebrafish ( Shiraki and Kawakami, 2018 Shiraki and Kawakami, 2018 ), mice ( Xu et al., 2017 Xu et al., 2017 ), and human cells ( Dong et al., 2017 Dong et al., 2017 ). Surprisingly, no obvious differences in efficiency were found between zebrafish tRNA and rice tRNA ( Shiraki and Kawakami, 2018 Shiraki and Kawakami, 2018 ). These findings demonstrated that the tRNA processing system is highly conserved in tested living organisms. Furthermore, the wide use of the PTG system is possible because of its non-toxicity ( Xu et al., 2017 Xu et al., 2017 ) and shorter sequence structure ( Mefferd et al., 2015 Mefferd et al., 2015 ). In this study, we also used the rice tRNA sequence to induce mutation of the medaka tyr gene in the embryos and cell line, and acquired the homozygote of tyr mutation by crossing and screening. We found that the PTG system did not show obvious toxicity for the embryogenesis of medaka, and the function of the tRNA processing system was also conserved between rice and medaka. Generally, gRNA is synthesized by in vitro transcription, or by in vivo transcription from an RNA polymerase III promoter such as U6 promoter ( Yin et al., 2015 Yin et al., 2015 ). gRNA synthesized by in vitro transcription has been demonstrated to induce gene editing in medaka ( Fang et al., 2018 Fang et al., 2018 ), but gRNA synthesized by in vitro transcription transfects fish cells only with difficulty, and degrades quickly in culture media ( Liu et al., 2018 Liu et al., 2018 ). Although human U6 can drive the transcription of gRNA to generate gene knockout in grass carp kidney cells (CIK) ( Ma et al., 2018 Ma et al., 2018 ), it does not work well in medaka cells ( Liu et al., 2018 Liu et al., 2018 ). Similarly, we have tried to express gRNA stably in vivo using RNA polymerase III promoters, such as medaka U6 promoter, medaka 7Sk promoter, or other U6 promoters from species like zebrafish, but these strategies were not successful in our previous experiments (data not shown). Interestingly and amazedly, the structure of gRNAs linked by tRNA can be processed into functional gRNAs under the control of CMV promoter or tissue-specific RNA polymerase II promoter in vivo ( Xu et al., 2017 Xu et al., 2017 ). Further, an improved tRNA scaffold has been engineered, which enables high gRNA production from RNA polymerase II promoters without ribozymes ( Knapp et al., 2019 Knapp et al., 2019 ). Here, we used CMV promoter, the most popular and strongest promoter yet found, to express gRNAs spaced by tRNA in vivo, and found mutation sequences in the tyr gRNA target site of both embryos and cells. Moreover, the designed vector pCS2-tRNA-PT containing Bbs I -Bbs I restriction enzyme sites is also suitable for cloning any candidate gRNA in one step, which is a robust system to generate functional gRNAs. The result in fact revealed that in vivo expression of PTG under the control of CMV promoter is completely feasible in medaka. The success of this experiment has inspired us to build a transgenic/mutated cell line and fish that stably express gRNA in vivo in the future. Although the SpCas9/tRNA-gRNA system was employed successfully in medaka, some shortcomings were also observed. The tRNA-linked transcript was cleaved at the 5'-end and 3'-end of tRNA by RNaseP and RNaseZ, respectively (Forster and Altman, 1990; Schiffer et al., 2002), which means that time is required to convert tRNA-gRNA to gRNA and form the Cas9/gRNA complex in vivo. During embryonic development, the target DNA edited by gRNA/Cas9 occurred at lower levels in embryos injected with tRNA-gRNA compared to the embryos injected with gRNA. A similar result was found in zebrafish, in which the ratio of heteroduplex bands in embryos injected with three gRNAs was significantly higher than that in embryos injected with the three tRNA-linked gRNAs ( Shiraki and Kawakami, 2018 Shiraki and Kawakami, 2018 ). In summary, we successfully combined the PTG system under CMV promoter with the CRISPR/Cas9 system to induce gene editing in a medaka fish model. This is the first evidence that fish cell lines can be edited by a vector system that contains the CMV promoter and tRNA-gRNA expression cassette, suggesting that the PTG system will be a valuable tool for application of the CRISPR/Cas9 system in medaka, and logically also be used in other fish embryos or cell lines. 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 https://doi.org/10.1007/978-1-62703-113-4_7 Cis-acting ribozymes for the production of RNA in vitro transcripts with defined 5' and 3' ends 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 https://doi.org/10.1105/tpc.16.00922 A multipurpose toolkit to enable advanced genome engineering in plants 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 https://doi.org/10.1038/s41598-017-00654-2 A convenient Cas9-based conditional knockout strategy for simultaneously targeting multiple genes in mouse 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 https://doi.org/10.1002/jez.b.22742 Identification and expression of conserved and novel RNA variants of medaka pax6b gene 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 https://doi.org/10.1016/j.bbrc.2016.11.129 Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells 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 https://doi.org/10.1002/jez.b.22808 Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9 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 https://doi.org/10.1631/jzus.B2000282 Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing 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 https://doi.org/10.1021/acssynbio.7b00259 Multiplexed CRISPR/Cas9 genome editing and gene regulation using Csy4 in Saccharomyces cerevisiae Forster AC , Altman S , 1990 . External guide sequences for an RNA enzyme . Science , 249 ( 4970 ): 783 - 786 . https://doi.org/10.1126/science.1697102 https://doi.org/10.1126/science.1697102 External guide sequences for an RNA enzyme 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 https://doi.org/10.1007/s00427-015-0493-4 Genetic interaction between Pax6 and β-catenin in the developing retinal pigment epithelium 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 https://doi.org/10.1016/j.jgg.2017.08.003 Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing 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 https://doi.org/10.1073/pnas.0308668101 Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro 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 https://doi.org/10.1073/pnas.1308335110 Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system 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 https://doi.org/10.1038/nbt.2508 RNA-guided editing of bacterial genomes using CRISPR-Cas systems 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 https://doi.org/10.1126/science.1225829 A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity 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 https://doi.org/10.1038/s41467-019-09148-3 Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression 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 https://doi.org/10.1631/jzus.B2100009 CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement 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 https://doi.org/10.1242/bio.035170 Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells 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 https://doi.org/10.1016/j.fsi.2018.02.039 Efficient resistance to grass carp reovirus infection in JAM-A knockout cells using CRISPR/Cas9 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 https://doi.org/10.1038/nmeth.2649 Cas9 as a versatile tool for engineering biology 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 https://doi.org/10.1261/rna.051631.115 Expression of CRISPR/Cas single guide RNAs using small tRNA promoters 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): e 55916. https://doi.org/10.3791/55916 https://doi.org/10.3791/55916 A protocol for multiple gene knockout in mouse small intestinal organoids using a CRISPR-concatemer 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 https://doi.org/10.1016/j.molcel.2014.04.022 Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells 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 https://doi.org/10.1016/j.cell.2014.09.014 CRISPR-Cas9 knockin mice for genome editing and cancer modeling 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 https://doi.org/10.1038/nmeth.3972 Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs 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 https://doi.org/10.1186/s12896-016-0289-2 High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize 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 https://doi.org/10.1038/cr.2015.95 Expansion of CRISPR/Cas9 genome targeting sites in zebrafish by Csy4-based RNA processing 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 https://doi.org/10.1038/nprot.2013.143 Genome engineering using the CRISPR-Cas9 system 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 https://doi.org/10.1093/emboj/21.11.2769 Assigning a function to a conserved group of proteins: the tRNA 3'-processing enzymes 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 https://doi.org/10.1038/s41598-018-31476-5 A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish 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 https://doi.org/10.1111/pbi.13390 Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining 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 https://doi.org/10.1631/jzus.B1900633 Gene editing: an instrument for practical application of gene biology to plant breeding 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 https://doi.org/10.1007/s00705-017-3553-4 CRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication Wang ZW , Zhu HP , Wang D , et al. , 2011 . A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp . BMC Res Notes , 4 : 82 . https://doi.org/10.1186/1756-0500-4-82 https://doi.org/10.1186/1756-0500-4-82 A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp 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 https://doi.org/10.1016/j.ymeth.2017.02.008 Gene editing in mouse zygotes using the CRISPR/Cas9 system 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 https://doi.org/10.1073/pnas.1420294112 Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system 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 https://doi.org/10.1007/s10529-015-1796-2 A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system 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 https://doi.org/10.1093/nar/gkw1048 Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA 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 https://doi.org/10.1111/jfd.12736 Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus 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 https://doi.org/10.1534/genetics.115.176917 Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs 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. 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). 10.1631/jzus.B2100343 1673-1581（2022）01-0074-10 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). 2021-04-11 2021-06-09 2022-01-15 2022-04-19 2021-12-09 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2021 2022 浙江大学出版社 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. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 1 23