A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elementsin vivo

Abstract

Human genetic studies have identified a large number of disease-associated de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects: the impact of regulatory variants is highly dependent on the cellular context, and thus for psychiatric diseases these would ideally be studied in neurons in a living brain. Furthermore, for both common and rare variants, it is expected that only a subset fraction will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with variants from ASD patients, we sought to develop a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. We implemented a Cre-dependent design to control expression of our library and first validated our system in vitro. Next, we measured the effect of >500 3′UTR variants in excitatory neurons in the mouse brain. Finally, we report >40 variants with significant effects on transcript abundance in the context of the brain. This new technique should enable robust, functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.

A Massively Parallel Screen of 5′UTR Mutations Identifies Variants Impacting Translation and Protein Production in Neurodevelopmental Disorder Genes

“…Our results in cell lines indicated that a subset of 5′UTR mutations from patients can alter protein production of host genes. However, regulation of translation can be cell type-specific (Cottrell et al, 2018; Lagunas et al, 2023), as certain factors such as the expression of helicases, initiation factors, and other RNA-binding proteins can vary between cellular contexts. Developing neurons are likely the most important cell type for autism (Fu et al, 2022; Li et al, 2023; Satterstrom et al, 2020).…”

“…Developing neurons are likely the most important cell type for autism (Fu et al, 2022; Li et al, 2023; Satterstrom et al, 2020). Therefore, we have previously adapted an MPRA for cell type-specific measures in neurons in vivo (Lagunas et al, 2023). Our previous approach was not sensitive enough to detect the effect of variants due to technical limitations of measuring small effect sizes with relatively few barcodes per reporter.…”

“…Finally, as autism is thought to be due to changes in neurons, we developed a new approach to assess the impact of allelic variants in neurons in the developing brain via MPRA. Building on our prior work where we had shown the ability to assess the effects of 3’ UTR elements (but not at that point alleles) in specific cell types in vivo (Lagunas et al, 2023) . To enable a more sensitive assay, we made several adaptations here.…”

Practical application of massively parallel reporter assay in biotechnology and medicine

“…Соматические мутации в 5'-нетранслируемых областях генов, которые потенциально влияют на эффективность трансляции, также обнаруживаются в клетках рака [164,165]. Использование МПРА позволило идентифицировать мутации в некодирую щих областях генов, ассоциированные с раком простаты [166], болезнями аутистического спект ра [167], а также с другими патологическими состоя ниями и признаками человека [98,168].…”

Rapid and Quantitative Functional Interrogation of Human Enhancer Variant Activity in Live Mice

“…Likewise, loss-of-function enhancer variants often result in loss of enhancer activity in one cell type, while in other cell types, its activity is unaffected (Bengani et al, 2021; Bhatia et al, 2015, 2021; Shin et al, 2023; Spieler et al, 2014). These cell-type-specific effects of enhancer variants are difficult to capture with high-throughput methods such as massively-parallel reporter assays (MPRAs) and CRISPR inhibitor/activator screens, both of which are primarily performed in vitro (Findlay, 2021; Inoue & Ahituv, 2015; Maricque et al, 2018) or in one tissue (Brown et al, 2022; Capauto et al, 2023; Deng et al, 2023; Lagunas et al, 2023; Patwardhan et al, 2012; White et al, 2013). Transgenic enhancer-reporter assays in mice enable visualization of enhancer activity in the whole animal and are a gold-standard for functionally testing when and where a human enhancer is active in vivo (Kothary et al, 1989; Pennacchio et al, 2006; Visel et al, 2006).…”