Shibata Y et al. (2022), CRISPR/Cas9-based simple transgenesis in Xenopu...

XB-ART-59158

Dev Biol 2022 Sep 01;489:76-83. doi: 10.1016/j.ydbio.2022.06.001.

Show Gene links Show Anatomy links

CRISPR/Cas9-based simple transgenesis in Xenopus laevis.

Shibata Y , Suzuki M , Hirose N , Takayama A , Sanbo C , Inoue T , Umesono Y , Agata K , Ueno N , Suzuki KT , Mochii M .

???displayArticle.abstract???
Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.

???displayArticle.pubmedLink??? 35690103
???displayArticle.link??? Dev Biol

Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: cga krt12.2 pam sox2 tgfbr2 tgfbr2l
GO keywords: CRISPR-cas system

???attribute.lit??? ???displayArticles.show???

	Graphical Abstract.
	Fig. 1. Generation of transgenic Xenopus with FGK promoter-dependent eGFP expression. (A) Schematic of Tg(NEXT-fgk:eGFP) integration at the tgfbr2l locus. (B) Phenotypes were classified into three groups depending on the fluorescent intensity and the localization: strong eGFP signal in the fin and gill including half transgenic animals; mosaic, weak eGFP signal only in the fin or gill; and none, no eGFP signals detected. Total and group sample numbers are indicated at the top and middle of each column, respectively. (C) Representative photographs of eGFP signals in Xla.Tg(NEXT-fgk:eGFP). Strong eGFP signals were detected in the fin and gill (a–c); weak eGFP signals were observed at the edge of the fin area (d); while no eGFP signals were detected in un-injected tadpoles (e). Arrowheads and double arrowheads indicate the gill and the edge of the tail fin, respectively. Bars = 1 mm. (D) Genotyping for Tg(NEXT-fgk:eGFP) integration in the tgfbr2l locus by PCR. PCR products were detected between 3.5 kb and 5 kb, suggesting Tg(NEXT-fgk:eGFP) was incorporated into the locus. Samples 1–3, strong eGFP signals were detected in the gill and the edge of the tail fin; samples 4–6, weak signals were only detected in the gill or edge of the tail fin; sample 7, un-injected tadpole. Detailed the electropherogram for the PCR products is indicated in Supplementary Fig. 7. HM: High molecular weight marker, LM: Low molecular weight marker and asterisk: primer dimer in TapeStation electropherogram.
	Fig. 2. Generation of transgenic Xenopus with CMV promoter-dependent tdTomato expression. (A) Schematic of Tg(NEXT-cmv:tdTomato) integration at the tgfbr2l locus. (B) Phenotypes were classified into three groups: strong tdTomato signals in the whole body including half transgenic animals; mosaic, weak tdTomato signals in the epithelial cells or muscle cells; and none, no tdTomato signals detected. Total and group sample numbers are indicated at the top and middle of each column, respectively. (C) Representative photographs of tdTomato signals in Xla.Tg(NEXT-cmv:tdTomato). Strong tdTomato signals were detected in the whole body (a, b) including half transgenic animals (d, e). c and f show bright field images of b and e, respectively. Bars = 1 mm. (D) Genotyping for Tg(NEXT-cmv:tdTomato) integration in the tgfbr2l locus by PCR. PCR products were detected between 3.5 kb and 5 kb, suggesting Tg(NEXT-cmv:tdTomato) was incorporated into the locus. Samples 1–3, strong tdTomato signals were detected in the gill and the edge of the tail fin; samples 4–6, weak signals were only detected in the gill or edge of the tail fin; sample 7, un-injected tadpole. Detailed the electropherogram for the PCR products is indicated in Supplementary Fig. 12. HM: High molecular weight marker, LM: Low molecular weight marker and asterisk: primer dimer in TapeStation electropherogram.
	Fig. 3. NEXTrans is applicable for the generation of various transgenic X. laevis. (A, B) Representative photographs of Xla.Tg(NEXT-cryga:tdTomato). Arrowheads indicating the presence or absence of a tdTomato signal in the eye. (A) and (B) show fluorescent and bright field merged images, respectively. (C, D, E) Representative photographs of Xla.Tg(NEXT-sox2:tdTomato). (D) and (E) show the high magnification of the region marked by a dotted line in (C). Abbreviations: Br, Brain; On, Optical nerve; Oln, Olfactory nerve; Olb, Olfactory bulb; Ey, Eye. Bars = 1 mm for (A–C), 0.5 mm for (D, E).
	Fig. 4. Germline transmission of various transgenic X. laevis generated by NEXTrans. (A) Representative photographs of F1 offspring in Xla.Tg(NEXT-fgk:egfp) (a–d), and Xla.Tg(NEXT-cryga:tdTomato) (e and f) embryos. Arrowheads and double arrowheads indicate the gill and the fin edge, respectively. b and d show bright field images of a and c, respectively. On the other hand, arrows show the eye with tdTomato signal. Bars = 1 mm. (B) The table summarizes the germline transmission rates of various transgenic animals. F1 embryos were generated by natural mating or in vitro fertilization outcrossed with wild type females. Two adult males were used for Xla.Tg(NEXT-fgk:egfp). The germline transmission rates were scored at stage 40–43.
	Supplementary Figure 1. Cas9 RNP allows highly efficient target gene mutagenesis in X. laevis. (A) sgRNA for both tyrosinase (tyr.L, tyr.S) is designed at conserved region in exon 1. (B) Representative photographs for uninjected tadpoles (a, b) and Cas9 RNP-injected tadpoles (c, d). Ten of thirteen (10/13) tyr sgRNA injected tadpoles lead to nearly complete albinism (c, d). Bars = 1 mm
	Supplementary Figure 2. Heteroduplex mobility assay for tgfbr2.l sgRNA1. (A) Genomic sequences of tgfbr2l.L. Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence which are inserted in the plasmid vectors. Blue letters indicate tgfbr2l.L exon 4. The light green highlighted sequence indicates sgRNA1. The bold indicates tgfbr2l.L HMA Forward primer sequence and the underlined indicate primer sequence for tgfbr2l.L HMA Reverse. (B) Genomic sequences of tgfbr2l.S. Blue letters indicate tgfbr2l.S exon 4. The light green highlighted sequence indicates sgRNA1. The bold indicates tgfbr2l.S HMA Forward primer sequence and the underlined indicate primer sequence for tgfbr2l.S HMA Reverse. (C) The expected homoduplex PCR product sizes (wt allele) were 481 bp or 477 bp for tgfbr2l.L or tgfbr2l.S, respectively. Slower paced migration of multiple bands, heteroduplex PCR products, due to annealing of wt and indel mutant alleles caused by sgRNA1 in PAGE were seen, suggesting that the sgRNA can recognize both gene and effectively induce mutation in both the target region.
	Supplementary Figure 3. Full plasmid sequence of Tg(NEXT-fgk:eGFP). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue and green letters indicate FGK promoter and eGFP sequence, respectively. The underlined indicates sgRNA1 sequence. Vector backbone is based on pEGFP-1 (Clontech, CA, USA) described in Suzuki et al (2010).
	Supplementary Figure 3. Full plasmid sequence of Tg(NEXT-fgk:eGFP). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue and green letters indicate FGK promoter and eGFP sequence, respectively. The underlined indicates sgRNA1 sequence. Vector backbone is based on pEGFP-1 (Clontech, CA, USA) described in Suzuki et al (2010).
	Supplementary Figure 4. Comparison of expression rates of transgene among three CRISPR sgRNAs. (A) Three sgRNAs are designed at the exon4 of tgfbr2l gene loci. Blue and bold letters indicate the sgRNAs sequence and PAM sequences in each, respectively. The red line shows the putative double strand break induced by Cas9 RNP. (B) The eGFP expression ratio induced by different types of sgRNA. eGFP+ includes embryos with mosaic and weak fluorescence. sgRNA1 and Tg(NEXT-fgk:eGFP) injection shoed a higher eGFP epression rate than the rates ith sgRNA2 and sgRNA3. Similar results were obtained from two independent experiments.
	Supplementary Figure 5. Primer design for detecting transgene at the tgfbr2l.L and tgfbr2l.S loci. (A, B) Genomic sequences of tgfbr2l.L and tgfbr2l.S, respectively. Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence which are inserted in the plasmid vectors. Blue letters indicate tgfbr2l.L and tgfbr2l.S exon 4. The light green highlighted and light blue highlighted sequence indicate sgRNA1 and sgRNA 3, respectively. The underlined showed sgRNA2 sequence. The bold letters and arrows indicate primer sequence for genotyping to detect transgene.
	Supplementary Figure 6. Comparison of genome sequences between tgfbr2l.L and tgfbr2l.S in the exon 4. Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence which are inserted in the plasmid vectors. The underlined showed sgRNA1 sequence. Since sgRNA1 sequence was designed to recognize in 100% matched region, Cas9 RNP was expected to induce double strand break in both the target site. The asterisks indicate the matched nucleotides. The 90.1% nucleotides were matched between tgfbr2l.L and tgfbr2l.S.
	Supplementary Figure 7. The electropherogram for the PCR product sizes of tgfbr2l.L forward insertion in Xla.Tg(NEXT-fgk: eGFP) tadpoles described in Figure 1D. The PCR products were detected between 3.5 kb and 5 kb with different peak size 4437 bp for sample 1, 4378 bp for sample 2 (including some minor products), 3390 bp and 4070 bp for sample 3, respectively. Since target plasmid vector integrated into the target locus via NonHomologous End Joining (NHEJ), the various indel mutations were supposed in both 5 and 3 junctions. Dashed squares and asterisks indicate the area between 3.5 kb and 5 kb and primer dimer peaked at around 60 bp. HM: Higher marker, LM: Lower marker.
	Supplementary Figure 8. Representative sequences of 5´ junction among tgfbr2l.L forward insertion in Xla.Tg(NEXT-fgk: eGFP) tadpoles. (A) Schematic model for 5´ junction between tgfbr2l.L locus and Tg(NEXTfgk:eGFP). (B) The 5´ junction sequence was amplified from the genomic DNA of Cas9 RNP and Tg(NEXTfgk:eGFP) injected tadpoles which showed strong eGFP signals in the fin and gill as described in Figure 1D. Clear single band was amplified in Number 1 and 3 transgenic tadpoles, whereas two bands were detected in Number 2 animals between 1 kb and 1.5 kb. L: Ladder. (C) PCR products were directly sequenced by using tgfbr2l.L Fw2 primer after purification. Deleted and inserted nucleotides in the DNA sequences are indicated by dashes and red letters, respectively. Bold letters and underlines show sgRNA1 sequences and PAM sequences, respectively.
	Supplementary Figure 9. Xla.Tg(NEXT-fgk: eGFP) tadpoles completed metamorphosis normally. (a)(b) Representative photographs of the Xla.Tg(NEXT-fgk:eGFP) tadpoles (mosaic) at stage 44. (c)(d) Representative photographs of the Xla.Tg(NEXT-fgk:eGFP) tadpoles (strong) at stage 56. (c) bright field, (d) GFP. (e-g) Representative photographs of Xla.Tg(NEXT-fgk:eGFP) froglets (strong) at stage 66 after metamorphosis. Ten transgenic animals completed metamorphosis with no developmental defect (N=10). The asterisk, arrowheads, and double arrowheads indicate the gill, eye, and the edge of the tail fin with eGFP signal, respectively. (a-d) Bars: 1 mm. (f)(g) Bars: 2 mm.
	Supplementary Figure 10. Full plasmid sequence of Tg(NEXT-cmv: tdTomato). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue and red letters indicate CMV promoter and tdTomato sequence, respectively. The underlined sequence indicates sgRNA1. Vector backbone is based on pEGFP-1 (Clontech) described in Suzuki et al. (2010). cmv:tdTomato cassette was cloned from ptdTomato-N1 (Clontech) using PCR and was modified.
	Supplementary Figure 10. Full plasmid sequence of Tg(NEXT-cmv: tdTomato). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue and red letters indicate CMV promoter and tdTomato sequence, respectively. The underlined sequence indicates sgRNA1. Vector backbone is based on pEGFP-1 (Clontech) described in Suzuki et al. (2010). cmv:tdTomato cassette was cloned from ptdTomato-N1 (Clontech) using PCR and was modified.
	Supplementary Figure 11. Representative photographs of Xla.Tg(NEXT-cmv:tdTomato) with mosaic or weak tdTomato signals. Weak or mosaic tdTomato signals were observed in the entire skin surface (a), epidermal and muscle cells (b)(c) in the part of the body. Bars = 1 mm.
	Supplementary Figure 12. The electropherogram for the PCR product sizes of tgfbr2l.L forward insertion in Xla.Tg(NEXT-cmv: tdTomato) tadpoles described in Figure 2D. Because of target plasmid integration via NHEJ causing indels, the PCR products were detected between 3.5 kb and 5 kb with different peak sizes, 3928 bp for sample 1, 3884 bp for sample 2, and 3905 bp for sample 3, respectively. Dashed squares and asterisks indicate the area between 3.5 kb and 5 kb and primer dimer peaked at around 60 bp. HM: Higher marker, LM: Lower marker.
	Supplementary Figure 13. Full plasmid sequence of Tg(NEXT-cryga:tdTomato). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue and red letters indicate gamma crystallin promoter and tdTomato sequence, respectively. The underlined sequence indicates sgRNA1. Vector backbone is based on pCMV-Tet3G (Clontech). cryga and tdTomato were cloned from Xenopus tropicalis genome DNA according to Offield et al. (2000) and pCMV-tdToamato, respectively and were modified.
	Supplementary Figure 13. Full plasmid sequence of Tg(NEXT-cryga:tdTomato). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue and red letters indicate gamma crystallin promoter and tdTomato sequence, respectively. The underlined sequence indicates sgRNA1. Vector backbone is based on pCMV-Tet3G (Clontech). cryga and tdTomato were cloned from Xenopus tropicalis genome DNA according to Offield et al. (2000) and pCMV-tdToamato, respectively and were modified.
	Supplementary Figure 14. Xla.Tg(NEXT-cryga:tdTomato) tadpoles completed metamorphosis normally. Representative photographs of the heads of Xla.Tg(NEXT-cryga:tdTomato) tadpoles at stage 56 (a)(b), and at stage 66 after metamorphosis (c-i). (a)(d)(g) tdTomato, (b)(f)(i) bright field with tdTomato, (c)(e)(h) bright field. (d-f) Half transgenesis (N=28). (g-i) Full transgenesis (N=12). Arrowheads indicate the eye with tdTomato signal. Bars: 2 mm.
	Supplementary Figure 15. Full plasmid sequence of Tg(NEXT-sox2:tdTomato). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue, green, and red letters indicate sox2 promoter, N2 enhancer fragment, and tdTomato sequence, respectively. The underlined sequence indicates sgRNA1. Vector backbone is based on pEGFP-1 (Clontech) described in Suzuki et al. (2010). sox2 promoter/N2 enhancer and tdTomato were cloned from Xenopus tropicalis genome DNA and pCMV-tdTomato (Clontech) using PCR, respectively and were modified.
	Supplementary Figure 15. Full plasmid sequence of Tg(NEXT-sox2:tdTomato). Yellow highlighted letters indicate 665 bp of tgfbr2l.L sequence. Blue, green, and red letters indicate sox2 promoter, N2 enhancer fragment, and tdTomato sequence, respectively. The underlined sequence indicates sgRNA1. Vector backbone is based on pEGFP-1 (Clontech) described in Suzuki et al. (2010). sox2 promoter/N2 enhancer and tdTomato were cloned from Xenopus tropicalis genome DNA and pCMV-tdTomato (Clontech) using PCR, respectively and were modified.
	Supplementary Figure 16. Representative photos of Xla.Tg(NEXT-sox2:tdTomato) with sox2 promoter dependent tdTomato signals. Representative photos for Xla.Tg(NEXT-sox2:tdTomato). (A) Dorsal view. The (i) and (ii) figures show the high magnification for square dotted line in (A). (B)(C) Lateral view. Br, Brain; On, Optical nerve; Oln, Olfactory nerve; Olb, Olfactory bulb; Ey, Eye. Ba, Branchial arches; Mc, Meckels cartilage; Cc, Ceratohyal cartilage. Bars = 1 mm for (A-C), 0.5 mm for (i) and (ii).
	Supplementary Figure 17. Representative photos of Xla.Tg(NEXT-cmv:tdTomato) with cmv promoter dependent tdTomato signals. (A) Representative photographs of F1 offspring in Xla.Tg(NEXT-cmv:tdTomato) embros. Top panel indicates the embros ith or ithout tdTomato signal and bottom shos bright field image of top panel. Bars = 1 mm. (B) The table summaries the germline transmission rate of the cmv transgenic animals. F1 embros ere generated b in vitro fertiliation outcrossed ith ild tpe females. The germline transmission rates ere scored at stage 40-43.
	Supplementary Figure 18. Genotyping for Tg(NEXT-fgk:egf) inegain in he gfb2 lci b PCR in F1 ibling. Genomic DNA as etracted from si Xla.Tg(NEXT-fgk:egfp) F1 siblings ith clear eGFP signals in the fin edge and gill and ild tpe embro. The PCR product as amplified using four site specific primers and common eGFP Reverse primer set, detailed information is described in Table S2, to detect the plasmid insertion in the tgfbr2l loci. All eGFP positive siblings ere observed plasmid insertion in the tgfbr2l loci, No. 1, 4-6: tgfbr2l.S forard direction, No. 2: tgfbr2l.L forard direction, and No. 3: tgfbr2l.L reverse direction. No plasmid insertion as detected in the tgfbr2l.S ith reverse direction. Since target plasmid vector integrated into the target loci via Non-Homologous End Joining (NHEJ), the various indel mutations were supposed in both 5 and 3 junctions. Yellow arrows indicate expected PCR products in each electrophoresis image. L: DNA ladder.
	Supplementary Figure 19. Genotyping for Tg(NEXT-cga:dTa) inegain in he gfb2 lci b PCR in F1 ibling. Ten Xla.Tg(NEXT-crga:tdTomato) F1 siblings ith tdTomato signals in both ees ere used for genotping PCR. The PCR product as amplified using tgfbr2l.S F and tdTomato Reverse primer set to detect the forard insertion in the tgfbr2l.S locus. No plasmid insertion as detected in the tgfbr2l.L locus ith both directions and in the tgfbr2l.S locus ith reverse direction (data not shon). Since target plasmid vector integrated into the target loci via Non-Homologous End Joining (NHEJ), the various indel mutations were supposed in both 5 and 3 junctions. Yellow arrow indicates expected PCR products. L: DNA ladder.