Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator‐like effector nucleases

Abstract

Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

Manipulation and <em>In Vitro</em> Maturation of <em>Xenopus laevis</em> Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

“…In addition, overexpression of chromatin modifying factors before fertilization may make it possible to remodel maternal chromatin before fertilization for understanding maternal chromatin states necessary for development. This strategy may also work well with current gene editing systems such as Transcription activator-like effector nuclease (TALEN) 18,19 and CRISPR/CAS9 20,21 since these can be expressed even before fertilization. Therefore, our new approach has a potential to be used for many applications in future.…”

“…Unused 10 mg/ml Digitonin can be frozen and stored at -20 °C and re-used) to 1 ml of the sperm solution. 19 The glass pipette has to be wide enough to accommodate oocytes without squeezing. 2.…”

Plant genome editing by novel tools: TALEN and other sequence specific nucleases

“…TALENs were recently used in a broad range of metazoans from simple organisms like Drosophila [21], Xenopus [22], Branchiostoma [23], and Zebrafish [24] to more complex vertebrates like mice [25], rats [26], pigs [27] and non-human primates [28]. In most of these organisms TALENs have been used to disrupt genes by NHEJ to study gene knockouts or establish the system in the organisms such as Branchiostoma [23], but human disease models can also be studied using TALENs.…”

CRISPR/Cas9: The Leading Edge of Genome Editing Technology

“…Kim et al ( 2014a ) applied the CRISPR/Cas9 system to the genotyping of polymorphisms in vitro. Restriction fragment length polymorphism (RFLP) analysis is often used for genotyping of genome-edited alleles (Suzuki et al 2013 ;Nakagawa et al 2014 ;Sakane et al 2014 ). However, conventional RFLP analysis can only be applied when there is a recognition sequence for a restriction enzyme around the DSB site.…”