Biology subjects

Sanchez, H. M.

Publications and source records attributed to Sanchez, H. M..

4 recordsLinked to original sources

bioRxiv2024

High-throughput optimized prime editing mediated endogenous protein tagging for pooled imaging of protein localization

The subcellular organization of proteins carries important information on cellular state and gene function, yet currently there are no technologies that enable accurate measurement of subcellular protein localizations at scale. Here we develop an approach for pooled endogenous protein tagging using prime editing, which coupled with an optical readout and sequencing, provides a snapshot of proteome organization in a manner akin to perturbation-based CRISPR screens. We constructed a pooled library of 17,280 pegRNAs designed to exhaustively tag 60 endogenous proteins spanning diverse localization patterns and explore a large space of genomic and pegRNA design parameters. Pooled measurements of tagging efficiency uncovered both genomic and pegRNA features associated with increased efficiency, including epigenetic states and interactions with transcription. We integrate pegRNA features into a computational model with predictive value for tagging efficiency to constrain the design space of pegRNAs for large-scale peptide knock-in. Lastly, we show that combining in-situ pegRNA sequencing with high-throughput deep learning image analysis, enables exploration of subcellular protein localization patterns for many proteins in parallel following a single pooled lentiviral transduction, setting the stage for scalable studies of proteome dynamics across cell types and environmental perturbations.

cell biology↗

bioRxiv2021

Eliminating Mosquitoes with Precision Guided Sterile Males

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and zika, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and eliminate mosquitoes in multigenerational population cages. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

genetics↗

bioRxiv2019

Vector bionomics and vectorial capacity as emergent properties of mosquito behaviors and ecology

Mosquitoes are important vectors for pathogens of humans and other vertebrate animals. Some aspects of adult mosquito behavior and mosquito ecology play an important role in determining the capacity of vector populations to transmit pathogens. Here, we re-examine factors affecting the transmission of pathogens by mosquitoes using a new approach. Unlike most previous models, this framework considers the behavioral states and state transitions of adult mosquitoes through a sequence of activity bouts. We developed a new framework for individual-based simulation models called MBITES (Mosquito Bout-based and Individual-based Transmission Ecology Simulator). In MBITES, it is possible to build models that simulate the behavior and ecology of adult mosquitoes in exquisite detail on complex resource landscapes generated by spatial point processes. We also developed an ordinary differential equation model which is the Kolmogorov forward equations for models developed in MBITES under a specific set of simplifying assumptions. While infection of the mosquito and pathogen development are one possible part of a mosquitos state, that is not the main focus. Using extensive simulation using some models developed in MBITES, we show that vectorial capacity can be understood as an emergent property of simple behavioral algorithms interacting with complex resource landscapes, and that relative density or sparsity of resources and the need to search can have profound consequences for mosquito populations capacity to transmit pathogens.

ecology↗

bioRxiv2018

Transforming Insect Population Control with Precision Guided Sterile Males

The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed \"precision guided SIT\" (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. To demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to transform our ability to control insect agricultural pests and disease vectors.

synthetic biology↗