Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.
Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.
Department of Life Systems, Institute of Technology and Science, University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima 770-8506, Japan.
Takahito
T
Watanabe
Hiroshi
H
Ochiai
Tetsushi
T
Sakuma
Hadley W
HW
Horch
Naoya
N
Hamaguchi
Taro
T
Nakamura
Tetsuya
T
Bando
Hideyo
H
Ohuchi
Takashi
T
Yamamoto
Sumihare
S
Noji
Taro
T
Mito
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques / Q000379:methods*; D049750:Genome, Insect; D006135:Gryllidae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D016335:Zinc Fingers
D019476:Insect Proteins; D003851:Deoxyribonucleases