The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

Abstract

Neuropsychiatric phenotypes have been long known to be influenced by heritable risk factors. The past decade of genetic studies have confirmed this directly, revealing specific common and rare genetic variants enriched in disease cohorts. However, the early hope for these studies-that only a small set of genes would be responsible for a given disorder-proved false. The picture that has emerged is far more complex: a given disorder may be influenced by myriad coding and noncoding variants of small effect size, and/or by rare but severe variants of large effect size, many de novo.Noncoding genomic sequences harbor a large portion of these variants, the molecular functions of which cannot usually be inferred from sequence alone. This creates a substantial barrier to understanding the higher-order molecular and biological systems underlying disease risk. Fortunately, a proliferation of genetic technologies-namely, scalable oligonucleotide synthesis, high-throughput RNA sequencing, CRISPR, and CRISPR derivatives-have opened novel avenues to experimentally identify biologically significant variants en masse. These advances have yielded an especially versatile technique adaptable to large-scale functional assays of variation in both untranscribed and untranslated regulatory features: Massively Parallel Reporter Assays (MPRAs). MPRAs are powerful molecular genetic tools that can be used to screen tens of thousands of predefined sequences for functional effects in a single experiment. This approach has several ideal features for psychiatric genetics, but remains underutilized in the field to date. To emphasize the opportunities MPRA holds for dissecting psychiatric polygenicity, we review here its applications in the literature, discuss its ability to test several biological variables implicated in psychiatric disorders, illustrate this flexibility with a proof-of-principle, in vivo cell-type specific implementation of the assay, and envision future outcomes of applying MPRA to both computational and experimental neurogenetics.

Systematic identification of cis-regulatory variants that cause gene expression differences in a yeast cross

“…Massively parallel reporter assays (MPRAs) have begun to make it possible to dissect regulatory activity of DNA sequences at scale ( Kinney et al, 2010 ; Melnikov et al, 2012 ; Mulvey et al, 2020 ; Patwardhan et al, 2009 ). In these approaches, which have been applied in bacteria ( Cambray et al, 2018 ; Kinney et al, 2010 ; Kosuri et al, 2013 ), yeast ( Cuperus et al, 2017 ; Mogno et al, 2013 ; Sharon et al, 2012 ), flies ( Gisselbrecht et al, 2013 ), zebrafish ( Rabani et al, 2017 ), mouse tissues ( Kwasnieski et al, 2014 ; Smith et al, 2013 ), and cultured human cells ( Mulvey et al, 2020 ), pooled libraries of DNA oligos are placed next to or in a reporter gene and inserted into populations of cells. The activity of each oligo is then assayed in bulk by pooled high-throughput sequencing.…”

Commercial Release of Genetically Modified Crops in Africa: Interface Between Biosafety Regulatory Systems and Varietal Release Systems

“…DNA-based methods involve genetic interaction mapping (Gonatopoulos-Pournatzis et al, 2020), DNA-protein interactions (Cherstvy et al, 2008), and DNA accessibility or DNA-binding assays. RNA level-based approaches include the use of microarrays (Plomin and Schalkwyk, 2007), RNA sequencing (RNA-seq), serial analysis of RNA expression (Yamamoto et al, 2001), and massively parallel reporter assays (Melnikov et al, 2014;Kehe et al, 2019;Mulvey et al, 2020). Protein level-based techniques include the yeast-two hybrid mutation (Schwartz et al, 1998;Kondo et al, 2003) and deep mutation scanning, among other methods.…”

Massively parallel identification of causal variants underlying gene expression differences in a yeast cross

“…Massively parallel reporter assays (MPRAs) have begun to make it possible to dissect regulatory activity of DNA sequences at scale (Kinney et al, 2010;Melnikov et al, 2012;Mulvey et al, 2020;Patwardhan et al, 2009). In these approaches, which have been applied in bacteria (Cambray et al, 2018;Kinney et al, 2010;Kosuri et al, 2013), yeast (Cuperus et al, 2017;Mogno et al, 2013;Sharon et al, 2012), flies (Gisselbrecht et al, 2013), zebrafish (Rabani et al, 2017), mouse tissues (Kwasnieski et al, 2014;Smith et al, 2013), and cultured human cells (Mulvey et al, 2020), pooled libraries of DNA oligos are placed next to or in a reporter gene and inserted into populations of cells. The activity of each oligo is then assayed in bulk by pooled high-throughput sequencing.…”