Repeat mediated excision of gene drive elements for restoring wild-type populations.

Here, we demonstrate that single strand annealing (SSA) can be co-opted for the precise autocatalytic excision of a drive element. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). By engineering direct repeats flanking the drive allele and inducing a double-strand DNA break (DSB) at a second endonuclease target site within the allele, we increased the utilization of SSA repair.

Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti.

While gene drive strategies have been proposed to aid in the control of mosquito-borne diseases, additional genome engineering technologies may be required to establish a defined end-of-product-life timeline. We previously demonstrated that single-strand annealing (SSA) was sufficient to program the scarless elimination of a transgene while restoring a disrupted gene in the disease vector mosquito Aedes aegypti. Here, we extend these findings by establishing that complete transgene removal (four gene cassettes comprising ~8-kb) can be programmed in cis.

The Rac1 homolog CED-10 is a component of the MES-1/SRC-1 pathway for asymmetric division of the EMS blastomere.

Asymmetric cell division is essential for the creation of cell types with different identities and functions. The EMS blastomere of the four-cell embryo undergoes an asymmetric division in response to partially redundant signaling pathways. One pathway involves a Wnt signal emanating from the neighboring P2 cell, while the other pathway is defined by the receptor-like MES-1 protein localized at the EMS/P2 cell contact, and the cytoplasmic kinase SRC-1.

Repeat mediated excision of gene drive elements for restoring wild-type populations.

We demonstrate here that single strand annealing (SSA) repair can be co-opted for the precise autocatalytic excision of a drive element. Although SSA is not the predominant form of DNA repair in eukaryotic organisms, we increased the likelihood of its use by engineering direct repeats at sites flanking the drive allele, and then introducing a double-strand DNA break (DSB) at a second endonuclease target site encoded within the drive allele. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE).

An knock-in allele for investigation of molting and oscillatory gene regulation.

NHR-85 is a poorly characterized nuclear hormone receptor transcription factor with an emerging role in regulating microRNA expression to control developmental timing. We generated the first NHR-85 translational fusion by knocking a cassette into the endogenous locus to tag all known isoforms. animals have wild-type broodsizes and NHR-85 ::GFP peaks in expression at the start of the L4 stage in epithelial cells.