Biology subjects

Anderson, M. A. E.

Publications and source records attributed to Anderson, M. A. E..

4 recordsLinked to original sources

bioRxiv2023

A synthetic biology approach to transgene expression

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most non-model and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3 untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and promoter sequences, representing a significant advancement in mosquito synthetic biology gene expression. tools. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/555539v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db954forg.highwire.dtl.DTLVardef@1279543org.highwire.dtl.DTLVardef@1b86fa2org.highwire.dtl.DTLVardef@1a9176a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

AePUb promoter length modulates gene expression in Aedes aegypti

Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects.

molecular biology↗

bioRxiv2023

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti

Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.

bioengineering↗

bioRxiv2022

A multiplexed, confinable CRISPR/Cas9 gene drive propagates in caged Aedes aegypti populations

Aedes aegypti, the yellow fever mosquito, is the main vector of several major pathogens including yellow fever, dengue, Zika and chikungunya viruses. Classical mosquito control strategies, mainly utilizing insecticides, have had success in controlling other mosquito vectors in recent years, but are much less useful against Ae. aegypti, and even these methods are threatened by rising insecticide resistance. This has stimulated interest in new mosquito control mechanisms, notably genetic systems such as gene drives. However, the development of CRISPR/Cas9 gene drive systems has faced challenges such as low inheritance biasing rate, the emergence of resistance alleles, and the possibility of spreading beyond the intended population. Here, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 in the germline to find an expression timing more conducive to homing. We also created a separate multiplexing (targeting multiple different sites within the target gene) sgRNA-expressing homing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene to limit the consequences of resistance alleles. This creates a split gene drive such that one part does not drive, allowing control over geographic spread and temporal persistence. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element was driven through a cage trial population such that carrier frequency of the element increased from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system. Significance statementMosquito-borne diseases affect millions of people worldwide, with the yellow fever mosquito (Aedes aegypti) being the principal vector of many viral diseases. Effective measures for controlling this mosquito are sorely needed. Gene drive systems have arisen as a potential tool for mosquito control due to their ability of biasing inheritance of a trait into a target population. Here, we assess a split gene drive, based on CRISPR/Cas9 endonuclease technology driving a target element into the mosquito population. Evaluated over successive generations in a replicated cage trial, the drive successfully biased its inheritance, increasing in frequency from 50% to up to 89%. Our results are encouraging for the potential use of this type of contained gene drive system for mosquito control in endemic areas.

synthetic biology↗