High‑efficiency system for construction and evaluation of customized TALENs for silkworm genome editing.
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.
Feng
F
Wang
Sanyuan
S
Ma
Hanfu
H
Xu
Jianping
J
Duan
Yuancheng
Y
Wang
Huan
H
Ding
Yuanyuan
Y
Liu
Xiaogang
X
Wang
Ping
P
Zhao
Qingyou
Q
Xia
D000818:Animals; D012831:Bombyx / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D004789:Enzyme Activation / Q000235:genetics; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049750:Genome, Insect; D016296:Mutagenesis / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D015203:Reproducibility of Results; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D014157:Transcription Factors; D003851:Deoxyribonucleases