Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis

Csy4-based vector system enables conditional chimeric gene editing in zebrafish without... Dear Editor, Despite diverse transfection approaches, zygote micro-injection remains the most widely used method to deliver the CRISPR/Cas9 system into most model organisms, including zebrafish and non-human primates (Jao et al., 2013; Niu et al., 2014). In zebrafish, injection of Cas9 mRNA and single guide RNA (sgRNA) is routine, and sgRNAs are usually produced via in vitro transcription driven by T7 promoter, which prefers 5′-GG as the initial nucleotide of transcripts, constraining the selection of target sites (Sanjana et al., 2014). Recently, several RNA processing mechanisms were employed to generate sgRNAs from cleavable multiplexed transcripts, which not only expand the available target sites but also empower the spatiotemporal regulation of sgRNA expression via injecting vectors carrying RNA polymerase II promoters (Nissim et al., 2014; Xu et al., 2017). Among those mechanisms, RNA endonuclease Csy4 simply requests a 28-nucleotide RNA sequence (‘28’) for recognition. However, a severe teratogenic effect was previously observed after injecting csy4 mRNA into zebrafish zygotes, prohibiting the further application of Csy4 in manipulating vertebrate embryos (Qin et al., 2015). Here we integrated csy4 with the CRISPR/Cas9 system into Tol2-flanked vectors (Kwan et al., 2007), and used conditional and tissue-specific promoters to regulate the expression of all components in vivo. Zebrafish zygotes injected by the cys4-expressing vectors could develop into viable chimeric embryos and larvae with a high frequency of integration, allowing for inducible gene editing without causing malformation. To determine whether the teratogenic effect can be attenuated by introducing Csy4 at later stages of embryogenesis, we generated two vectors: one carrying cas9 and csy4 driven by the hsp70l promoter, and the other only containing cas9 after the hsp70l promoter (Supplementary Figure S1A). Both vectors were flanked by Tol2 transposon ends, and 30 pg of each vector was co-injected into zebrafish zygotes with 30 pg tol2 mRNA, followed by one-time heat shock between 2 and 8 h post-fertilization (hpf). All embryos were examined at 24 hpf, and most embryos that were injected with the hsp70l:cas9-2a-csy4 vector and heat-shocked after 6 hpf maintained normal morphology (Supplementary Figure S1B–D). Although there is no evidence that the zebrafish genome harbors Csy4-binding sequence, transcription analysis suggested that expression of several homeotic genes (hox genes) at 10 hpf was deregulated after introducing Csy4 at 2 hpf (Supplementary Figure S1E). Next, we generated another Tol2-flanked vector containing hsp70l promoter followed by a DNA template of egfp sgRNA flanked by two Csy4-binding sites (Figure 1A). The hsp70l:28-sgRNA-28 vector (20 pg) was co-injected into heterozygous Tg(krt4:eGFP) embryos together with the hsp70l:cas9-2a-csy4 vector (30 pg) and tol2 mRNA (30 pg), and the embryos were heat-shocked twice at 8 hpf and 16 hpf. At 3.5 dpf, while control Tg(krt4:eGFP) larvae were covered by a uniform layer of keratinocytes expressing the green fluorescent protein (GFP), the injected larvae displayed variable mosaic fluorescent patterns due to the chimeric knockout of egfp (Figure 1B and Supplementary Figure S3A), confirming the successful cleavage of an effective egfp sgRNA from the fused transcript. Figure 1 View largeDownload slide Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis. (A) Schematic diagram of the hsp70l:cas9-2a-csy4 vector and hsp70l:28-sgRNA-28 vector. (B) Lateral views of a normal 3.5 dpf krt4:eGFP larva (top) and a 3.5 dpf krt4:eGFP larva with egfp partially knocked out (bottom). Scale bar, 250 μm. (C) (i) Schematic diagram of the optimized knock-in donor vector. (ii) Representative images of injected krt4:eGFP larvae carrying a few keratinocytes with mKate2 knocked in. Scale bar, 30 μm. (D) (i) Schematic diagram of the hsp70l:28-sgRNA-28 vector integrated by a universal tracking cascade ef1α:mKate2. (ii) Representative images of injected krt4:eGFP (top) and 1016α1tubulin:eGFP (bottom) larvae. The green and red fluorescence was distributed in a complementary way in keratinocytes and midbrain neurons, respectively. Scale bar, 12 μm. (E) (i) Schematic diagram of the hsp70l:28-sgRNA1-28-28-sgRNA2-28 vector integrated by a myocardium-specific tracking cascade myl7:eGFP. (ii) Representative images of hearts of a normal myl7:eGFP larva (left) and an injected larva with strong tracing fluorescence in the heart region (right). (iii) Statistics of chamber size of atrium and ventricle at diastole and systole stages in both normal hearts and the hearts with tnnt2a partially disrupted (n = 8 per group). Scale bar, 50 μm. (F) (i) Schematic diagram of tre:mKate2-triplex-28-sgRNA-28 and tre:cas9-2a-csy4 vectors, as well as the transgenic zebrafish model fabp10a:teton; tre:eGFP-krasv12. The liver-specific expression of mKate2 and CRISPR/Cas9/Csy4 system in injected larvae is activated in the presence of doxycycline, and subsequently disrupts the oncogenic eGFP-Krasv12. (ii) Illustration and representative images of wild-type liver (left), tumorizing liver upon eGFP-Krasv12 insult (middle), and tumorizing liver with eGFP-Krasv12 partially knocked out (right). (iii) Statistics of liver size (n = 8 per group). Scale bar, 200 μm. Mean ± SEM, with statistical differences determined by one-way analysis of variance. *P < 0.05, **P < 0.005. Figure 1 View largeDownload slide Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis. (A) Schematic diagram of the hsp70l:cas9-2a-csy4 vector and hsp70l:28-sgRNA-28 vector. (B) Lateral views of a normal 3.5 dpf krt4:eGFP larva (top) and a 3.5 dpf krt4:eGFP larva with egfp partially knocked out (bottom). Scale bar, 250 μm. (C) (i) Schematic diagram of the optimized knock-in donor vector. (ii) Representative images of injected krt4:eGFP larvae carrying a few keratinocytes with mKate2 knocked in. Scale bar, 30 μm. (D) (i) Schematic diagram of the hsp70l:28-sgRNA-28 vector integrated by a universal tracking cascade ef1α:mKate2. (ii) Representative images of injected krt4:eGFP (top) and 1016α1tubulin:eGFP (bottom) larvae. The green and red fluorescence was distributed in a complementary way in keratinocytes and midbrain neurons, respectively. Scale bar, 12 μm. (E) (i) Schematic diagram of the hsp70l:28-sgRNA1-28-28-sgRNA2-28 vector integrated by a myocardium-specific tracking cascade myl7:eGFP. (ii) Representative images of hearts of a normal myl7:eGFP larva (left) and an injected larva with strong tracing fluorescence in the heart region (right). (iii) Statistics of chamber size of atrium and ventricle at diastole and systole stages in both normal hearts and the hearts with tnnt2a partially disrupted (n = 8 per group). Scale bar, 50 μm. (F) (i) Schematic diagram of tre:mKate2-triplex-28-sgRNA-28 and tre:cas9-2a-csy4 vectors, as well as the transgenic zebrafish model fabp10a:teton; tre:eGFP-krasv12. The liver-specific expression of mKate2 and CRISPR/Cas9/Csy4 system in injected larvae is activated in the presence of doxycycline, and subsequently disrupts the oncogenic eGFP-Krasv12. (ii) Illustration and representative images of wild-type liver (left), tumorizing liver upon eGFP-Krasv12 insult (middle), and tumorizing liver with eGFP-Krasv12 partially knocked out (right). (iii) Statistics of liver size (n = 8 per group). Scale bar, 200 μm. Mean ± SEM, with statistical differences determined by one-way analysis of variance. *P < 0.05, **P < 0.005. For comparison of efficiency, we knocked out the same egfp target site by injecting several alternative combinations (Supplementary Figure S1F). The combinations consisted of two or three vectors and RNAs in the following list: cas9 mRNA (300 pg), sgRNA (30 pg), Tol2-flanked zu6:sgRNA vector (20 pg), hsp70l:cas9-2a-csy4 vector (30 pg), hsp70l:28-sgRNA-28 vector (20 pg), and tol2 mRNA (30 pg). All injected Tg(krt4:eGFP) embryos were subjected to two heat shocks at 8 hpf and 16 hpf, and the knockout efficiency was evaluated by the proportion of GFP-negative skin area from lateral view (Supplementary Figure S1F and G). Additionally, a target site on gdf11 gene was also used to test the knockout efficiency of different combinations by assessing the cleavage ratio in T7E1 assay (Supplementary Figure S2A–C). Statistics from both experiments showed that the injection of Tol2-flanked Cas9/Csy4-based vectors achieved comparable knockout efficiency as the conventional pure RNA injection (Supplementary Figures S1F and S2C). In addition, to improve the knockout efficiency, we also generated a Tol2-flanked vector expressing a fused transcript containing double sgRNAs targeting two adjacent sites on the egfp sequence (one starts with GG and the other starts with AG). The double sgRNA vector performed significantly better than both single sgRNA vectors using the same injection dosage (30 pg) (Supplementary Figure S4A–C). Subsequently, the Cas9/Csy4-based vector system was tested for non-homologous end joining (NHEJ) knock-in assay. Here, we aimed to replace the eGFP in heterozygous Tg(krt4:eGFP) with a red fluorescent protein mKate2. To avoid the effect of the potential frame shift, we modified the donor by inserting three stop codes separated by a single nucleotide (TGACTAACTAG) after the bait sequence, followed by an internal ribosome entry site (IRES) element and a Gal4/UAS-mediated mKate2 expression cascade (Figure 1C; Supplementary Figures S3B and S5A). The donor vector (15 pg) was then co-injected with hsp70l:28-sgRNA-28 vector (20 pg), hsp70l:cas9-2a-csy4 vector (30 pg), and tol2 mRNA (30 pg). After heat shocks, we observed only a few keratinocytes switching to red fluorescence, whereas most cells just lost green fluorescence (Figure 1C). Therefore, in comparison to the conventional RNA-based protocol (Supplementary Figure S5B), Cas9/Csy4-based vector system did not work well for targeted knock-in via zygote injection, probably due to the fast dilution of donor vectors during the early embryogenesis. Since many cells in injected F0 embryos and larvae carried expressions from Tol2 constructs (Supplementary Figure S2D), we tested several different tracking strategies to help trace cells that harbor transgenes, and therefore, may bear disrupted alleles. We first included a tracking cascade expressing mKate2 driven by the ef1α promoter into the hsp70l:28-sgRNA-28 vector, and all somatic cells with integrated CRISPR/Cas9/Csy4 system were constitutively labeled by red fluorescence (Figure 1D and Supplementary Figure S3A). The vector was co-injected with the hsp70l:cas9-2a-csy4 vector into heterozygous Tg(krt4:eGFP) or heterozygous Tg(1016α1tubulin:eGFP), in which keratinocytes or neurons expressed GFP. The injected embryos were heat-shocked at different stages to induce the expression of CRISPR/Cas9/Csy4, and the green fluorescence gradually faded or disappeared from the keratinocytes and neurons labeled in red fluorescence (Figure 1D), indicating effective knockout of egfp in the traced cells. Second, we integrated a tracking cascade expressing GFP driven by the myl7 promoter into a vector expressing double sgRNAs targeting tnnt2a (Figure 1E; Supplementary Figures S3C and S4D). This tracking cascade allowed us to identify larvae with more myocardium-specific integration by observing the intensity of green fluorescence in the heart region, and we found that the cardiac chambers were statistically enlarged with weaker systole in the selected larvae compared to the wild-type control (Figure 1E). Since only the homologous tnnt2a mutant displayed cardiac contraction defects at larval stages (Liu et al., 2017), it was speculated that both alleles of tnnt2a gene may be simultaneously knocked out in some traced cardiocytes. Lastly, we employed the inducible Tet-On system to perform tissue-specific tracking and gene knockout. For demonstration, Tg(fabp10a:teton;tre:eGFP-krasv12) was utilized as a zebrafish model of liver cancer, in which the liver-specific expression of the GFP-fused oncogene krasv12 was triggered by doxycycline administration, and led to subsequent hyperplasia and tumorigenesis (Figure 1F and Supplementary Figure S3A). To disrupt the trans-oncogene, we generated Tol2-flanked vectors expressing both Cas9-2A-Csy4 and 28-sgRNA-28 cascades via the tre promoter, and an mkate2 sequence followed by an 110-bp stabilizing triplex sequence was placed right upstream of 28-sgRNA-28 (Figure 1F) (Nissim et al., 2014). The vectors and tol2 mRNA were co-injected into zebrafish zygotes, and doxycycline was treated from 2.5 dpf. At 6 dpf, larvae with significant red fluorescence in the liver area were selected for analysis. As expected, the hepatocytes labeled in red fluorescence gradually lost the expression of GFP-Krasv12, and the size of the tumorizing liver with trans-oncogene partially knocked out was significantly smaller than that of the un-injected tumorizing liver (Figure 1F). In addition, the BrdU assay further confirmed that the proliferation of oncogenic hepatocytes was greatly reduced upon oncogene knockout, with no alteration in the rate of apoptosis (Supplementary Figure S4C and D). Previously, to achieve tissue-specific or inducible gene editing in zebrafish, several groups have attempted to deliver the CRISPR/Cas9 system into F0 embryos via vectors using either a zu6 promoter to produce sgRNAs, or ribozyme-mediated sgRNAs expression (Ablain et al., 2015; Lee et al., 2016). Compared with zu6 promoters, Csy4-based vector system allows us to regulate the expression patterning, timing, and dosage of sgRNAs. Compared with self-cleaving ribozymes, Csy4-mediated cleavage requires the expression of an additional protein, and seems less convenient. However, the unique mechanism of Csy4-mediated cleavage improves the diversity of synthetic biology toolbox for manipulating RNA sequence, and provides alternative spatiotemporal control for designing sophisticated regulatory circuits (Nissim et al., 2014). Together, here we provide evidence that zygote injection of the Csy4-based vector system can realize flexible regulations of RNA processing without inducing significant malformation, and therefore, Csy4 may be reconsidered as a useful component in programming complex synthetic networks not only in vitro but also in vivo for precisely manipulating the genome of model organisms. [Supplementary material is available at Journal of Molecular Cell Biology online. This study was supported by the Foundation for Returned Oversea Chinese Scholars (48-12), the National Natural Science Foundation of China (81402582), and Natural Science Foundation of Shanghai (14YF1400600).] References Ablain , J. , Durand , E.M. , Yang , S. , et al. . ( 2015 ). A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish . Dev. Cell 32 , 756 – 764 . Google Scholar CrossRef Search ADS PubMed Jao , L.E. , Wente , S.R. , and Chen , W. ( 2013 ). Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system . Proc. Natl Acad. Sci. USA 110 , 13904 – 13909 . Google Scholar CrossRef Search ADS Kwan , K.M. , Fujimoto , E. , Grabher , C. , et al. . ( 2007 ). The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs . Dev. Dyn. 236 , 3088 – 3099 . Google Scholar CrossRef Search ADS PubMed Lee , R.T. , Ng , A.S. , and Ingham , P.W. ( 2016 ). Ribozyme mediated gRNA generation for in vitro and in vivo CRISPR/Cas9 mutagenesis . PLoS One 11 , e0166020 . Google Scholar CrossRef Search ADS PubMed Liu , L. , Zhang , R.R. , Yang , Q. , et al. . ( 2017 ). Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis . Sheng Li Xue Bao 69 , 267 – 275 . Google Scholar PubMed Nissim , L. , Perli , S.D. , 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 , 698 – 710 . Google Scholar CrossRef Search ADS PubMed Niu , Y. , Shen , B. , Cui , Y. , et al. . ( 2014 ). Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos . Cell 156 , 836 – 843 . Google Scholar CrossRef Search ADS PubMed 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 , 1074 – 1077 . Google Scholar CrossRef Search ADS PubMed Sanjana , N.E. , Shalem , O. , and Zhang , F. ( 2014 ). Improved vectors and genome-wide libraries for CRISPR screening . Nat. Methods 11 , 783 – 784 . Google Scholar CrossRef Search ADS PubMed Xu , L. , Zhao , L. , Gao , Y. , et al. . ( 2017 ). Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA . Nucleic Acids Res. 45 , e28 . Google Scholar PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis

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Abstract

Dear Editor, Despite diverse transfection approaches, zygote micro-injection remains the most widely used method to deliver the CRISPR/Cas9 system into most model organisms, including zebrafish and non-human primates (Jao et al., 2013; Niu et al., 2014). In zebrafish, injection of Cas9 mRNA and single guide RNA (sgRNA) is routine, and sgRNAs are usually produced via in vitro transcription driven by T7 promoter, which prefers 5′-GG as the initial nucleotide of transcripts, constraining the selection of target sites (Sanjana et al., 2014). Recently, several RNA processing mechanisms were employed to generate sgRNAs from cleavable multiplexed transcripts, which not only expand the available target sites but also empower the spatiotemporal regulation of sgRNA expression via injecting vectors carrying RNA polymerase II promoters (Nissim et al., 2014; Xu et al., 2017). Among those mechanisms, RNA endonuclease Csy4 simply requests a 28-nucleotide RNA sequence (‘28’) for recognition. However, a severe teratogenic effect was previously observed after injecting csy4 mRNA into zebrafish zygotes, prohibiting the further application of Csy4 in manipulating vertebrate embryos (Qin et al., 2015). Here we integrated csy4 with the CRISPR/Cas9 system into Tol2-flanked vectors (Kwan et al., 2007), and used conditional and tissue-specific promoters to regulate the expression of all components in vivo. Zebrafish zygotes injected by the cys4-expressing vectors could develop into viable chimeric embryos and larvae with a high frequency of integration, allowing for inducible gene editing without causing malformation. To determine whether the teratogenic effect can be attenuated by introducing Csy4 at later stages of embryogenesis, we generated two vectors: one carrying cas9 and csy4 driven by the hsp70l promoter, and the other only containing cas9 after the hsp70l promoter (Supplementary Figure S1A). Both vectors were flanked by Tol2 transposon ends, and 30 pg of each vector was co-injected into zebrafish zygotes with 30 pg tol2 mRNA, followed by one-time heat shock between 2 and 8 h post-fertilization (hpf). All embryos were examined at 24 hpf, and most embryos that were injected with the hsp70l:cas9-2a-csy4 vector and heat-shocked after 6 hpf maintained normal morphology (Supplementary Figure S1B–D). Although there is no evidence that the zebrafish genome harbors Csy4-binding sequence, transcription analysis suggested that expression of several homeotic genes (hox genes) at 10 hpf was deregulated after introducing Csy4 at 2 hpf (Supplementary Figure S1E). Next, we generated another Tol2-flanked vector containing hsp70l promoter followed by a DNA template of egfp sgRNA flanked by two Csy4-binding sites (Figure 1A). The hsp70l:28-sgRNA-28 vector (20 pg) was co-injected into heterozygous Tg(krt4:eGFP) embryos together with the hsp70l:cas9-2a-csy4 vector (30 pg) and tol2 mRNA (30 pg), and the embryos were heat-shocked twice at 8 hpf and 16 hpf. At 3.5 dpf, while control Tg(krt4:eGFP) larvae were covered by a uniform layer of keratinocytes expressing the green fluorescent protein (GFP), the injected larvae displayed variable mosaic fluorescent patterns due to the chimeric knockout of egfp (Figure 1B and Supplementary Figure S3A), confirming the successful cleavage of an effective egfp sgRNA from the fused transcript. Figure 1 View largeDownload slide Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis. (A) Schematic diagram of the hsp70l:cas9-2a-csy4 vector and hsp70l:28-sgRNA-28 vector. (B) Lateral views of a normal 3.5 dpf krt4:eGFP larva (top) and a 3.5 dpf krt4:eGFP larva with egfp partially knocked out (bottom). Scale bar, 250 μm. (C) (i) Schematic diagram of the optimized knock-in donor vector. (ii) Representative images of injected krt4:eGFP larvae carrying a few keratinocytes with mKate2 knocked in. Scale bar, 30 μm. (D) (i) Schematic diagram of the hsp70l:28-sgRNA-28 vector integrated by a universal tracking cascade ef1α:mKate2. (ii) Representative images of injected krt4:eGFP (top) and 1016α1tubulin:eGFP (bottom) larvae. The green and red fluorescence was distributed in a complementary way in keratinocytes and midbrain neurons, respectively. Scale bar, 12 μm. (E) (i) Schematic diagram of the hsp70l:28-sgRNA1-28-28-sgRNA2-28 vector integrated by a myocardium-specific tracking cascade myl7:eGFP. (ii) Representative images of hearts of a normal myl7:eGFP larva (left) and an injected larva with strong tracing fluorescence in the heart region (right). (iii) Statistics of chamber size of atrium and ventricle at diastole and systole stages in both normal hearts and the hearts with tnnt2a partially disrupted (n = 8 per group). Scale bar, 50 μm. (F) (i) Schematic diagram of tre:mKate2-triplex-28-sgRNA-28 and tre:cas9-2a-csy4 vectors, as well as the transgenic zebrafish model fabp10a:teton; tre:eGFP-krasv12. The liver-specific expression of mKate2 and CRISPR/Cas9/Csy4 system in injected larvae is activated in the presence of doxycycline, and subsequently disrupts the oncogenic eGFP-Krasv12. (ii) Illustration and representative images of wild-type liver (left), tumorizing liver upon eGFP-Krasv12 insult (middle), and tumorizing liver with eGFP-Krasv12 partially knocked out (right). (iii) Statistics of liver size (n = 8 per group). Scale bar, 200 μm. Mean ± SEM, with statistical differences determined by one-way analysis of variance. *P < 0.05, **P < 0.005. Figure 1 View largeDownload slide Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis. (A) Schematic diagram of the hsp70l:cas9-2a-csy4 vector and hsp70l:28-sgRNA-28 vector. (B) Lateral views of a normal 3.5 dpf krt4:eGFP larva (top) and a 3.5 dpf krt4:eGFP larva with egfp partially knocked out (bottom). Scale bar, 250 μm. (C) (i) Schematic diagram of the optimized knock-in donor vector. (ii) Representative images of injected krt4:eGFP larvae carrying a few keratinocytes with mKate2 knocked in. Scale bar, 30 μm. (D) (i) Schematic diagram of the hsp70l:28-sgRNA-28 vector integrated by a universal tracking cascade ef1α:mKate2. (ii) Representative images of injected krt4:eGFP (top) and 1016α1tubulin:eGFP (bottom) larvae. The green and red fluorescence was distributed in a complementary way in keratinocytes and midbrain neurons, respectively. Scale bar, 12 μm. (E) (i) Schematic diagram of the hsp70l:28-sgRNA1-28-28-sgRNA2-28 vector integrated by a myocardium-specific tracking cascade myl7:eGFP. (ii) Representative images of hearts of a normal myl7:eGFP larva (left) and an injected larva with strong tracing fluorescence in the heart region (right). (iii) Statistics of chamber size of atrium and ventricle at diastole and systole stages in both normal hearts and the hearts with tnnt2a partially disrupted (n = 8 per group). Scale bar, 50 μm. (F) (i) Schematic diagram of tre:mKate2-triplex-28-sgRNA-28 and tre:cas9-2a-csy4 vectors, as well as the transgenic zebrafish model fabp10a:teton; tre:eGFP-krasv12. The liver-specific expression of mKate2 and CRISPR/Cas9/Csy4 system in injected larvae is activated in the presence of doxycycline, and subsequently disrupts the oncogenic eGFP-Krasv12. (ii) Illustration and representative images of wild-type liver (left), tumorizing liver upon eGFP-Krasv12 insult (middle), and tumorizing liver with eGFP-Krasv12 partially knocked out (right). (iii) Statistics of liver size (n = 8 per group). Scale bar, 200 μm. Mean ± SEM, with statistical differences determined by one-way analysis of variance. *P < 0.05, **P < 0.005. For comparison of efficiency, we knocked out the same egfp target site by injecting several alternative combinations (Supplementary Figure S1F). The combinations consisted of two or three vectors and RNAs in the following list: cas9 mRNA (300 pg), sgRNA (30 pg), Tol2-flanked zu6:sgRNA vector (20 pg), hsp70l:cas9-2a-csy4 vector (30 pg), hsp70l:28-sgRNA-28 vector (20 pg), and tol2 mRNA (30 pg). All injected Tg(krt4:eGFP) embryos were subjected to two heat shocks at 8 hpf and 16 hpf, and the knockout efficiency was evaluated by the proportion of GFP-negative skin area from lateral view (Supplementary Figure S1F and G). Additionally, a target site on gdf11 gene was also used to test the knockout efficiency of different combinations by assessing the cleavage ratio in T7E1 assay (Supplementary Figure S2A–C). Statistics from both experiments showed that the injection of Tol2-flanked Cas9/Csy4-based vectors achieved comparable knockout efficiency as the conventional pure RNA injection (Supplementary Figures S1F and S2C). In addition, to improve the knockout efficiency, we also generated a Tol2-flanked vector expressing a fused transcript containing double sgRNAs targeting two adjacent sites on the egfp sequence (one starts with GG and the other starts with AG). The double sgRNA vector performed significantly better than both single sgRNA vectors using the same injection dosage (30 pg) (Supplementary Figure S4A–C). Subsequently, the Cas9/Csy4-based vector system was tested for non-homologous end joining (NHEJ) knock-in assay. Here, we aimed to replace the eGFP in heterozygous Tg(krt4:eGFP) with a red fluorescent protein mKate2. To avoid the effect of the potential frame shift, we modified the donor by inserting three stop codes separated by a single nucleotide (TGACTAACTAG) after the bait sequence, followed by an internal ribosome entry site (IRES) element and a Gal4/UAS-mediated mKate2 expression cascade (Figure 1C; Supplementary Figures S3B and S5A). The donor vector (15 pg) was then co-injected with hsp70l:28-sgRNA-28 vector (20 pg), hsp70l:cas9-2a-csy4 vector (30 pg), and tol2 mRNA (30 pg). After heat shocks, we observed only a few keratinocytes switching to red fluorescence, whereas most cells just lost green fluorescence (Figure 1C). Therefore, in comparison to the conventional RNA-based protocol (Supplementary Figure S5B), Cas9/Csy4-based vector system did not work well for targeted knock-in via zygote injection, probably due to the fast dilution of donor vectors during the early embryogenesis. Since many cells in injected F0 embryos and larvae carried expressions from Tol2 constructs (Supplementary Figure S2D), we tested several different tracking strategies to help trace cells that harbor transgenes, and therefore, may bear disrupted alleles. We first included a tracking cascade expressing mKate2 driven by the ef1α promoter into the hsp70l:28-sgRNA-28 vector, and all somatic cells with integrated CRISPR/Cas9/Csy4 system were constitutively labeled by red fluorescence (Figure 1D and Supplementary Figure S3A). The vector was co-injected with the hsp70l:cas9-2a-csy4 vector into heterozygous Tg(krt4:eGFP) or heterozygous Tg(1016α1tubulin:eGFP), in which keratinocytes or neurons expressed GFP. The injected embryos were heat-shocked at different stages to induce the expression of CRISPR/Cas9/Csy4, and the green fluorescence gradually faded or disappeared from the keratinocytes and neurons labeled in red fluorescence (Figure 1D), indicating effective knockout of egfp in the traced cells. Second, we integrated a tracking cascade expressing GFP driven by the myl7 promoter into a vector expressing double sgRNAs targeting tnnt2a (Figure 1E; Supplementary Figures S3C and S4D). This tracking cascade allowed us to identify larvae with more myocardium-specific integration by observing the intensity of green fluorescence in the heart region, and we found that the cardiac chambers were statistically enlarged with weaker systole in the selected larvae compared to the wild-type control (Figure 1E). Since only the homologous tnnt2a mutant displayed cardiac contraction defects at larval stages (Liu et al., 2017), it was speculated that both alleles of tnnt2a gene may be simultaneously knocked out in some traced cardiocytes. Lastly, we employed the inducible Tet-On system to perform tissue-specific tracking and gene knockout. For demonstration, Tg(fabp10a:teton;tre:eGFP-krasv12) was utilized as a zebrafish model of liver cancer, in which the liver-specific expression of the GFP-fused oncogene krasv12 was triggered by doxycycline administration, and led to subsequent hyperplasia and tumorigenesis (Figure 1F and Supplementary Figure S3A). To disrupt the trans-oncogene, we generated Tol2-flanked vectors expressing both Cas9-2A-Csy4 and 28-sgRNA-28 cascades via the tre promoter, and an mkate2 sequence followed by an 110-bp stabilizing triplex sequence was placed right upstream of 28-sgRNA-28 (Figure 1F) (Nissim et al., 2014). The vectors and tol2 mRNA were co-injected into zebrafish zygotes, and doxycycline was treated from 2.5 dpf. At 6 dpf, larvae with significant red fluorescence in the liver area were selected for analysis. As expected, the hepatocytes labeled in red fluorescence gradually lost the expression of GFP-Krasv12, and the size of the tumorizing liver with trans-oncogene partially knocked out was significantly smaller than that of the un-injected tumorizing liver (Figure 1F). In addition, the BrdU assay further confirmed that the proliferation of oncogenic hepatocytes was greatly reduced upon oncogene knockout, with no alteration in the rate of apoptosis (Supplementary Figure S4C and D). Previously, to achieve tissue-specific or inducible gene editing in zebrafish, several groups have attempted to deliver the CRISPR/Cas9 system into F0 embryos via vectors using either a zu6 promoter to produce sgRNAs, or ribozyme-mediated sgRNAs expression (Ablain et al., 2015; Lee et al., 2016). Compared with zu6 promoters, Csy4-based vector system allows us to regulate the expression patterning, timing, and dosage of sgRNAs. Compared with self-cleaving ribozymes, Csy4-mediated cleavage requires the expression of an additional protein, and seems less convenient. However, the unique mechanism of Csy4-mediated cleavage improves the diversity of synthetic biology toolbox for manipulating RNA sequence, and provides alternative spatiotemporal control for designing sophisticated regulatory circuits (Nissim et al., 2014). Together, here we provide evidence that zygote injection of the Csy4-based vector system can realize flexible regulations of RNA processing without inducing significant malformation, and therefore, Csy4 may be reconsidered as a useful component in programming complex synthetic networks not only in vitro but also in vivo for precisely manipulating the genome of model organisms. [Supplementary material is available at Journal of Molecular Cell Biology online. This study was supported by the Foundation for Returned Oversea Chinese Scholars (48-12), the National Natural Science Foundation of China (81402582), and Natural Science Foundation of Shanghai (14YF1400600).] References Ablain , J. , Durand , E.M. , Yang , S. , et al. . ( 2015 ). A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish . Dev. Cell 32 , 756 – 764 . Google Scholar CrossRef Search ADS PubMed Jao , L.E. , Wente , S.R. , and Chen , W. ( 2013 ). Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system . Proc. Natl Acad. Sci. USA 110 , 13904 – 13909 . Google Scholar CrossRef Search ADS Kwan , K.M. , Fujimoto , E. , Grabher , C. , et al. . ( 2007 ). The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs . Dev. Dyn. 236 , 3088 – 3099 . Google Scholar CrossRef Search ADS PubMed Lee , R.T. , Ng , A.S. , and Ingham , P.W. ( 2016 ). Ribozyme mediated gRNA generation for in vitro and in vivo CRISPR/Cas9 mutagenesis . PLoS One 11 , e0166020 . Google Scholar CrossRef Search ADS PubMed Liu , L. , Zhang , R.R. , Yang , Q. , et al. . ( 2017 ). Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis . Sheng Li Xue Bao 69 , 267 – 275 . Google Scholar PubMed Nissim , L. , Perli , S.D. , 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 , 698 – 710 . Google Scholar CrossRef Search ADS PubMed Niu , Y. , Shen , B. , Cui , Y. , et al. . ( 2014 ). Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos . Cell 156 , 836 – 843 . Google Scholar CrossRef Search ADS PubMed 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 , 1074 – 1077 . Google Scholar CrossRef Search ADS PubMed Sanjana , N.E. , Shalem , O. , and Zhang , F. ( 2014 ). Improved vectors and genome-wide libraries for CRISPR screening . Nat. Methods 11 , 783 – 784 . Google Scholar CrossRef Search ADS PubMed Xu , L. , Zhao , L. , Gao , Y. , et al. . ( 2017 ). Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA . Nucleic Acids Res. 45 , e28 . Google Scholar PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Molecular Cell BiologyOxford University Press

Published: Mar 21, 2018

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