indel-detection-following-crisprcas9-mutagenesis-using-high

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito <em>Aedes aegypti</em>

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

Texas A&M University

Aedes aegypti is a critical vector for transmitting Zika, dengue, chikungunya, and yellow fever viruses to humans. Genetic strategies to limit mosquito survival based upon sex distortion or disruption of development may be valuable new tools to control Ae. aegypti populations. We identified six genes with expression limited to pupal development; osi8 and osi11 (Osiris protein family), CPRs and CPF (cuticle protein family), and stretchin (a muscle protein). Heritable CRISPR/Cas9-mediated gene knockout of these genes did not reveal any defects in pupal development. However, stretchin-null mutations (strn) resulted in flightless mosquitoes with an abnormal open wing posture. The inability of adult strn mosquitoes to fly restricted their escape from aquatic rearing media following eclosion, and substantially reduced adult survival rates. Transgenic strains which contain the EGFP marker gene under the control of strn regulatory regions (0.8 kb, 1.4 kb, and 2.2 kb upstream, respectively), revealed the gene expression pattern of strn in muscle-like tissues in the thorax during late morphogenesis from L larvae to young adults. We demonstrated that Ae. aegypti pupae-specific strn is critical for adult mosquito flight capability and a key late-acting lethal target for mosquito-borne disease control.

A knockout screen of genes expressed specifically in Ae. aegypti pupae reveals a critical role for stretchin in mosquito flight.

Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3'-untranslated regions (3'UTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3'UTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3'UTR sequence, and tested their ability to contribute to the expression of reporter genes  or . Transient transfection in mosquito cells identified nine candidate 3'UTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3'UTR. Both synthetic 3'UTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3'UTR, albeit with differences in intensity. Thus, the synthetic 3'UTR elements described here are suitable for regulating transgene expression in , and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3'UTR elements in this or other organisms.

Emma Jakes

Department of Entomology and Agrilife Research

Texas A&M University

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Emma Jakes

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Entomology and Agrilife Research

Texas A&M University

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Department of Entomology and Agrilife Research