Suzuki KT et al. (2013), High efficiency TALENs enable F0 functional ana...

XB-ART-47296

Biol Open 2013 May 15;25:448-52. doi: 10.1242/bio.20133855.

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High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos.

Suzuki KT , Isoyama Y , Kashiwagi K , Sakuma T , Ochiai H , Sakamoto N , Furuno N , Kashiwagi A , Yamamoto T .

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Recently, gene editing with transcription activator-like effector nucleases (TALENs) has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr) and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.

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Species referenced: Xenopus laevis
Genes referenced: pax6 tyr

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	Fig. 1. Disruption of tyrosinase (tyr) in TALEN mRNA-injected embryos.(A) Phenotypes of tyr TALEN-injected embryos: Uninjected control embryos; TALEN-R, embryos injected with 1100âpg right tyr TALEN mRNA; TALEN-R/L, embryos injected with 550âpg of each right and left tyr TALEN mRNA. Injected embryos were reared to the hatching stage. (B) Detection of tyr mutations by genomic PCR and digestion with a restriction enzyme. Five embryos each were collected from the three experimental groups (Uninjected, TALEN-R, and TALEN-R/L). Upper image: a schematic drawing of tyr genomic PCR product. Genomic DNA of each embryo was prepared and then was subjected to PCR with a specific primer set for amplification containing the tyr TALEN target site. HinfI site is located on spacer sequence of the TALEN site. Middle image: a gel electrophoresis image of tyr genomic PCR products. Lower image: HinfI-digestion of PCR products for detecting tyr mutations. PCR products were purified and then were digested by HinfI. HinfI fragments are divided into 131 and 119âbp. M means 100âbp ladder marker. (C) Sequences and frequencies of tyr mutations. PCR product from the TALEN-R/L-injected embryo (asterisk in panel A) was subcloned and was subjected to DNA sequencing analysis. Mutant sequences were aligned to that of wild type. Sequence of wild-type allele is shown at the top. Yellow boxes indicate tyr TALEN target site. Gaps generated by deletion are shown as blue dashes. A substitution is shown as an underlined blue character. Types and frequencies of each mutation (deletion and insertion) are shown at the right of the panel.
	Fig. 2. Phenotypes of tyr TALEN-injected embryos.Embryos were divided into four groups according to their phenotypes. (A) Representative phenotypes: Severe, near complete loss of pigmentation in retina pigmented epithelium (RPE); Moderate, more than half loss of pigmentation in RPE; Weak, less than half loss of pigmentation in RPE; Normal, no alteration of pigmentation. (B) Percentage of phenotypes in the three experimental groups, Uninjected, TALEN-R, and TALEN-R/L. Total numbers of individuals in this experiment are shown at the top of each graph.
	Fig. 3. Disruption of pax6 in the TALEN mRNA-injected embryos.(A) Phenotypes of pax6 TALEN-injected embryos: TALEN-R, embryos injected with 1100âpg right pax6 TALEN mRNA; TALEN-R/L, embryos injected with 550âpg of each right and left pax6 TALEN mRNA. Injected embryos were reared to the hatching stage. Upper image: low magnification of each experimental group. Lower image: high magnification of representative phenotypes from TALEN-R- and TALEN-R/L-injected embryos noted by squares in the upper image. (B) Detection of pax6 mutations by genomic PCR and digestion with a restriction enzyme. One embryo was collected from each of the three experimental groups (asterisks in panel A): Uninjected, TALEN-R, and TALEN-R/L. Upper image: a schematic drawing of pax6 genomic PCR product. Genomic DNA of each embryo was prepared and then was subjected to PCR with a specific primer set for amplification containing the pax6 TALEN target site. Hpy188I site is located on spacer sequence of the TALEN site. Left image: a gel electrophoresis image of pax6 genomic PCR products. Right image: Hpy188I-digestion of PCR products for detecting pax6 mutations. PCR products were purified and then digested by Hpy188I. Hpy188I fragments are divided into 101 and 54âbp. M means 100âbp ladder marker. Uninjected indicates uninjected control embryo. (C) Sequences and frequencies of pax6 mutation by TALENs. PCR product from the TALEN-R/L-injected embryo (asterisk in panel A) was subcloned and was subjected to DNA sequencing analysis. Mutant sequences were aligned to those of wild type. Sequences of pax6a and pax6b wild-type alleles are shown at the top. Yellow boxes indicate pax6 TALEN target site. Gaps generated by deletion are shown as blue dashes. Types and frequencies of each mutation (deletion and insertion) are shown at the right of the panel. Paralogous substitutions between pax6a and pax6b are highlighted in green.
	Fig. 4. Phenotypes of pax6 TALEN-injected embryos.Embryos were divided into four groups according to their phenotypes. (A) Representative phenotypes: Severe, near complete loss of eyes; Eye deformation, embryos with deformed eyes; Small eyes, embryos with apparently small eyes as compared with the Uninjected and TALEN-R-injected embryos; Normal, no alteration of eye formation and size. (B) Percentage of phenotypes in the three experimental groups, Uninjected, TALEN-R, and TALEN-R/L. Total numbers of individuals in this experiment are shown at the top of each graph.

References [+] :

Cade, Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. 2012, Pubmed