Shibata Y et al. (2023), Protocols for transgenesis at a safe harbor sit...

XB-ART-60157

STAR Protoc 2023 Sep 15;43:102382. doi: 10.1016/j.xpro.2023.102382.

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Protocols for transgenesis at a safe harbor site in the Xenopus laevis genome using CRISPR-Cas9.

Shibata Y , Okumura A , Mochii M , Suzuki KT .

Abstract
We have established a new transgenesis protocol based on CRISPR-Cas9, "New and Easy XenopusTransgenesis (NEXTrans)," and identified a novel safe harbor site in African clawed frogs, Xenopus laevis. We describe steps in detail for the construction of NEXTrans plasmid and guide RNA, CRISPR-Cas9-mediated NEXTrans plasmid integration into the locus, and its validation by genomic PCR. This improved strategy allows us to simply generate transgenic animals that stably express the transgene. For complete details on the use and execution of this protocol, please refer to Shibata et al. (2022).1.

PubMed ID: 37389994
PMC ID: PMC10511863
Article link: STAR Protoc

Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: krt12.2 sox2 tgfbr2l

Article Images: [+] show captions

	Graphical abstract
	Figure 1. Schematic comparison of the principle of transgenesis using REMI, I-SceI, and NEXTrans methods Transgenic plasmids are randomly incorporated at multiple loci in the Xenopus laevis genome by the restriction enzyme-mediated integration (REMI)2 and meganuclease (I-SceI)-mediated3 transgenesis methods, whereas the NEXTrans plasmid with CRISPR-Cas9 is integrated into a novel safe harbor site, the tgfbr2l locus.
	Figure 2. Schematic representation of NEXTrans (New and Easy Xenopus Transgenesis) at a novel harbor site The NEXTrans plasmid carrying the transgene cassette is co-injected into fertilized Xenopus laevis eggs with Cas9 RNP targeting the tgfbr2l.L and tgfbr2l.S loci (exon 4). The plasmid integrates into the target sites in the zygotic genome in a forward and/or reverse direction by NHEJ repair during the early embryonic stage and can be detected by PCR. Primer pairs were used for PCR genotyping to detect the Tg(NEXT-fgk:egfp) integration as follows: primers 1 and 5 for tgfbr2l.L forward insertion, primers 2 and 5 for tgfbr2l.L reverse insertion, primers 3 and 5 for tgfbr2l.S forward insertion, and primers 4 and 5 for tgfbr2l.S reverse direction (see key resources table).
	Figure 3. Representative transgenic X. laevis founders generated by NEXTrans (A) Representative photograph of eGFP signals in Xla.Tg(NEXT-fgk:egfp) in a promoter/enhancer manner. Strong eGFP signals were detected in the fin and gill. (B) Mosaic transgenic Xla.Tg(NEXT-fgk:egfp) embryo expressing eGFP only in the fin edge. (C) Representative photograph of ubiquitous tdTomato signals in Xla.Tg(NEXT-cmv:tdtomato). Strong tdTomato signals were detected in the whole body. (D) Half-transgenic Xla.Tg(NEXT-cmv:tdtomato) embryo expressing tdTomato signals only on one side of the body. (E) Representative photograph of tdTomato signals in Xla.Tg(NEXT-cryga:tdtomato) in a promoter/enhancer manner. (F) Fully transgenic Xla.Tg(NEXT-cryga:tdtomato) with tdTomato signals detected in both eyes completed the metamorphosis normally. Arrows indicate tdTomato signals in the eye; arrowheads indicate the gill; double arrowheads indicate the edge of the tail fin. Scale bars = 1 mm for (A–E) and 5 mm for (F).
	Figure 4. Representative PCR genotyping for targeted integration of NEXTrans plasmid PCR genotyping was performed using the primer pairs described in Figure 2 and key resources table to detect Tg(NEXT-fgk:egfp) integration in tgfbr2l loci. The genomic DNA was extracted from F1 siblings (#2) generated by in vitro fertilization. Green arrowhead indicates expected PCR products, suggesting Tg(NEXT-fgk:egfp) was integrated into the tgfbr2l.S locus in the forward direction. Samples 1–4, strong eGFP signals were detected in the gill and the edge of the tail fin; sample 5, wild-type (WT) tadpole. HM, high molecular weight marker; LM, low molecular weight marker in the TapeStation electropherogram.
	Figure 5. Germline transmission of transgenic X. laevis generated by NEXTrans Representative photographs of F1 offspring of (A) Xla.Tg(NEXT-fgk:egfp), (B) Xla.Tg(NEXT-cmv:tdtomato), and (C) Xla.Tg(NEXT-cryga:tdtomato) embryos. Arrowheads indicate the gill; double arrowheads indicate the edge of the tail fin; arrow indicates the eye with tdTomato signal. Scale bars = 1 mm.

References [+] :

Auer, Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. 2014, Pubmed