An efficient TALEN mutagenesis system in rice

Kunling Chen, Qiwei Shan, Caixia Gao

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5months.

Original languageEnglish (US)
Pages (from-to)2-8
Number of pages7
JournalMethods
Volume69
Issue number1
DOIs
StatePublished - 2014

Bibliographical note

Funding Information:
This work was supported by the Ministry of Agriculture of China ( 2013ZX08002-004 and 2013ZX08010-002 ), and the National Natural Science Foundation of China ( 201263 , 383601 and 31200273 ).

Keywords

  • Gene knockout
  • Rice
  • TALENs
  • Targeted mutagenesis

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