The CRISPR/Cas System mediates efficient genome engineering in Bombyx mori

Wang, Yueqiang; Li, Zhiqian; Xu, Jun; Zeng, Baosheng; Lin, Ling; You, Lei; Chen, Yazhou; Huang, Yongping; Tan, Anjiang

doi:10.1038/cr.2013.146

Cited by 259 publications

(233 citation statements)

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“…CRISPR/Cas9-mediated Knock-out of E75B in Bombyx-Our colleagues previously developed efficient approaches for CRISPR/Cas9-mediated genome editing in Bombyx (37,38) and helped us to perform the E75B knock-out experiment in this study. Cas9 mRNA (mMESSAGE mMACHINE kit, Ambion, Austin, TX) and E75B sgRNA (MAXIscript T7 Kit, Ambion, Austin, TX), TAATACGACTCACTATAGGTGCT-AGTGAGCATGCTGGAGGGTTTTAGAGCTAGAAATAG-CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA-AAAGTGGCACCGAGTCGGTGCTTTT, was synthesized and purified separately.…”

Section: Methodsmentioning

confidence: 99%

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx

Tian

Guo

et al. 2016

Journal of Biological Chemistry

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The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing.Animals undergo developmental transitions from the embryo to juvenile to adulthood, and these processes are determined by steroid hormones and their corresponding nuclear receptors (NRs).2 In insects, 20-hydroxyecdysone (20E; ecdysone is the immediate precursor of 20E; 20E and ecdysone are the main ecdysteroids) is the actual steroid hormone. The ecdysone receptor (EcR) and its partner molecule, Ultraspiracle (USP), form the functional NR complex of 20E. In conjunction with EcR-USP, 20E activates a small set of early response genes encoding several transcription factors that further activate a large set of downstream late response genes. Pulses of 20E signals initiate major developmental transitions in insects, including egg hatching, larval-larval molting, and larval-pupal-adult metamorphosis (1, 2). NRs form a large and conserved superfamily of ligand-activated transcription factors that are essential for growth, development, reproduction, homeostasis, and metabolism. NRs are defined by the presence of a highly conserved DNA binding domain (DBD) and a less conserved ligand binding domain (3, 4). There are 18 -19 NRs in insects, including the fruit fly, Drosophila melanogaster, and the silkworm, Bombyx mori (3,5,6). Apart from the EcR, ligand was only identified for another insect NR, ecdysone-induced protein 75B (E75). E75 is a crucial 20E response gene that affects ecdysteroid titer. E75 binds to heme, which responds to gases . The E75 orthologs in mammals are Rev-erb ␣ (NR1D1) and Rev-erb ␤ (NR1D2), and NR1D2 binds to heme, responds to NO, and regulates circadian rhythm (13-15).In Drosophila, the E75 locus encodes four E75 mRNA isoforms, E75A, E75B, E75C, and E75D, which are generated by differential promoter usage and alternative splicing of 5Ј exons. The DBD of E75A/C possesses two C4 zinc fingers; E75B is incomplete and contains only one zinc finger, whereas E75D lacks a DBD. 20E-...

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Section: Methodsmentioning

confidence: 99%

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx

Tian

Guo

et al. 2016

Journal of Biological Chemistry

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“…The earliest published reports of Cas9-mediated genome editing in Lepidoptera, from 2013 and 2014, all describe work done in B. mori (Wang et al 2013;Ma et al 2014;Wei et al 2014) -an experimental system that benefits from a large research community that had already developed efficient methods for injection, rearing, and genotyping. To our knowledge, Wang et al (2013) represent the first published report of Cas9-mediated genome editing in Lepidoptera and set three important precedents. First, they established the protocol that has been more or less emulated by most following studies, where single-guide RNAs (sgRNAs) are co-injected with Cas9 mRNA into early-stage embryos.…”

Section: Published Examples Of Cas9-mediated Genome Editing In Lepidomentioning

confidence: 99%

“…The first example of Cas9 genome editing in a species besides B. mori was described by Li et al (2015a), who produced deletions in three genes in the swallowtail butterfly Papilio xuthus. This was an important case study because it showed that the general approach used by Wang et al (2013) in B. mori could be transferred to other species and still retain the same level of high efficiency. Two more notable technical advancements include the production of an 18 kb deletion in B. mori by Zhang et al (2015) the longest deletion we know of in Lepidoptera, and much longer than anything in (Zhang and Reed 2016;Perry et al 2016), which was an important improvement to the protocol that significantly simplifies the genome editing workflow.…”

Section: Published Examples Of Cas9-mediated Genome Editing In Lepidomentioning

confidence: 99%

“…Third, they showed that deletions could occur in the germ line at a high enough frequency to generate stable lines. After Wang et al (2013), one of the next most important technical advancements came from Ma et al (2014), who showed that knockins could be achieved using a donor plasmid to insert a DsRed expression cassette using~1 kb homology arms. Following this, Zhu et al showed successful epitope tagging of BmTUDOR-SN gene by CRISPR/Cas9-mediated knockin in Bombyx cells (Zhu et al 2015).…”

Section: Published Examples Of Cas9-mediated Genome Editing In Lepidomentioning

confidence: 99%

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A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

Zhang

Reed

2017

Diversity and Evolution of Butterfly Wing Patterns

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CRISPR/Cas9 genome editing has revolutionized functional genetic work in many organisms and is having an especially strong impact in emerging model systems. Here we summarize recent advances in applying CRISPR/Cas9 methods in Lepidoptera, with a focus on providing practical advice on the entire process of genome editing from experimental design through to genotyping. We also describe successful targeted GFP knockins that we have achieved in butterflies. Finally, we provide a complete, detailed protocol for producing targeted long deletions in butterflies.

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“…Originally isolated from "CRISPR" acquired immune systems in bacteria, Cas9 is a non-repetitive enzyme that can be directed to cut almost any DNA sequence by simply expressing a "guide RNA" containing that same sequence. In little more than a year following the first demonstrations in human cells, it has enabled gene insertion, deletion, and replacement in many different species [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] . Building RNA-guided gene drives based on the Cas9 nuclease is a logical way to overcome the targeting and stability problems hindering gene drive development.…”

Section: Introductionmentioning

confidence: 99%

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

Esvelt

Smidler

Catteruccia

et al. 2014

Preprint

210

388

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Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

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The CRISPR/Cas System mediates efficient genome engineering in Bombyx mori

Cited by 259 publications

References 9 publications

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

Concerning RNA-Guided Gene Drives for the Alteration of Wild Populations

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