Molecular Genetics and Genomics

, Volume 288, Issue 12, pp 683–690

High-efficiency system for construction and evaluation of customized TALENs for silkworm genome editing

  • Feng Wang
  • Sanyuan Ma
  • Hanfu Xu
  • Jianping Duan
  • Yuancheng Wang
  • Huan Ding
  • Yuanyuan Liu
  • Xiaogang Wang
  • Ping Zhao
  • Qingyou Xia
Original Paper

DOI: 10.1007/s00438-013-0782-4

Cite this article as:
Wang, F., Ma, S., Xu, H. et al. Mol Genet Genomics (2013) 288: 683. doi:10.1007/s00438-013-0782-4

Abstract

Transcription activator-like effector nuclease (TALEN) possesses the characteristics of ease design and precise DNA targeting. In the silkworm Bombyx mori, TALEN has been successfully used to knockout an endogenous Bombyx gene, and shown the huge potential in functional genes research and improvement of the economical characteristics of silkworm. Thus, there is an urgent need to develop an applicable system that permits the efficient construction of customized TALEN with high activity that could efficiently induce the hereditable mutagenesis in the silkworm. In this study, we constructed an efficient assembly and evaluation system of the customized TALEN especiallly for silkworm genome editing by combination of a modified Golden Gate ligation strategy, a luciferase (LUC) reporter system in insect cell culture for binding activity and a surveyor nuclease assay system in silkworm embryos for cleavage efficiency. We showed the reliability of this system by assembling a pair of TALENs targeting a silkworm genome locus and assaying their binding and cleavage activities. The assembly strategy was convenient and efficient which allows the rapid construction of customized TALEN in less than 1 week, and the evaluation system was reliable and necessary for screening of the customized TALEN pair with high binding and cleavage activities. The results showed this system is a reliable and efficient tool for the construction of customized TALEN with high activity for gene targeting of silkworm, and will contribute to the wide application of TALEN technology in the functional gene research of silkworm.

Keywords

Transcription activator-like factorTALENsGenome editingSilkworm

Supplementary material

438_2013_782_MOESM1_ESM.pdf (211 kb)
Fig. S1. Strategy for assembly of TALENs with different length of repeats. Each strategy will generate one monomer library allowing for assembling many TALENs with same length of repeat. (PDF 211 kb)
438_2013_782_MOESM2_ESM.pdf (290 kb)
Fig. S2. PCR and repeat monomer sets used to generate monomer library. The position of each monomer and the primer sets used for PCR reaction are showed in the each wells. (PDF 289 kb)
438_2013_782_MOESM3_ESM.doc (39 kb)
Supplementary material 3 (DOC 39 kb)
438_2013_782_MOESM4_ESM.doc (48 kb)
Supplementary material 4 (DOC 47 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Feng Wang
    • 1
  • Sanyuan Ma
    • 1
  • Hanfu Xu
    • 1
  • Jianping Duan
    • 1
  • Yuancheng Wang
    • 1
  • Huan Ding
    • 1
  • Yuanyuan Liu
    • 1
  • Xiaogang Wang
    • 1
  • Ping Zhao
    • 1
  • Qingyou Xia
    • 2
  1. 1.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina
  2. 2.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina