Dataset Information

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

ABSTRACT: Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

SUBMITTER: Kojin BB

PROVIDER: S-EPMC9272899 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The global incidences of dengue virus have increased the interest in studying and understanding the mosquito population dynamics. It is predominantly spread by Aedes aegypti in the tropical and sub-tropical countries in the world. Understanding these dynamics is important for public health in countries where climatic and environmental conditions are favorable for the propagation of these diseases. For this reason, a new model has been proposed to investigate the population dynamics of mosquitoes in a city.The present paper discusses the numerical modeling of population dynamics of Ae. aegypti mosquitoes in an urban neighborhood of a city using the finite volume method. The model describes how populations spread through the city assisted by the wind. This model allows incorporating external factors (wind and chemical insecticides) and topography data (streets, building blocks, parks, forests and beach). The proposed model has been successfully tested in examples involving two Brazilian cities (City center, Juiz de Fora and Copacabana Beach, Rio de Janeiro).Invasion phenomena of Ae. aegypti mosquitoes have been observed in each of the simulations. It was observed that, inside the blocks, the growth of the population for both winged and aquatic phase causes an infestation of Ae. aegypti in a short time. Within the blocks the mosquito population was concentrated and diffused slowly. In the streets, there was a long-distance spread, which was influenced by wind and diffusion with a low concentration of mosquito population. The model was also tested taking into account chemical insecticides spread in two different configurations. It has been observed that the insecticides have a significant effect on the mosquito population for both winged and aquatic phases when the chemical insecticides spread more uniformly along all the streets in a neighborhood of a city.The presented methodology can be employed to evaluate and to understand the epidemic risks in a specific region of the city. Moreover the model allows an increase in efficiency of the existing mosquito population control techniques and to theoretically test new methods before involving the human population.

Dataset Information

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

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