Matthew A. Lalli

Background: Deleterious mutations in hundreds of genes confer high risk for neurodevelopmental disorders (NDDs), posing significant challenges for therapeutic development. Identifying convergent pathways shared across NDD genes could reveal high-impact therapeutic targets. Methods: To identity convergent pathways in NDD genes, we optimized Perturb-seq, a method combining CRISPR perturbation with single-cell RNA sequencing (scRNA-seq), and applied structural topic modeling (STM) to simultaneously assess impact on cell fate and developmental stage. We then studied a subset of autism spectrum disorder (ASD) genes implicated in regulation of gene expression using these improved molecular and analytical approaches. Results: Results from targeting 60 high-confidence ASD risk genes revealed significant effects on neural development. As expected, ASD risk genes impacted both progenitor fate and/or neuronal differentiation. Using STM, we could identify latent topics jointly capturing cell types, cell fate, and differentiation stages. Repression of ASD risk genes led to changes in topic proportions and effects of four genes ( DEAF1 , KMT2A , MED13L , and MYT1L) were validated in an independent dataset. Conclusions: Our optimized Perturb-seq method, combined with a novel analytical approach, provides a powerful, cost-effective framework for uncovering convergent mechanisms among genes involved in complex neurodevelopmental processes. Application of these methods advanced understanding of the impact of ASD mutations on multiple dimensions of neural development, and provides a framework for a broader examination of the function of NDD risk genes.

Calling cards technology using self-reporting transposons enables the identification of DNA–protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2 and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. Lastly, we demonstrated barcoded calling cards can record binding in vivo in the mouse brain. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells.

Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantified base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence GABAergic neurons, and oligodendrocytes. We found more selective editing and RNA hyper-editing in neurons relative to oligodendrocytes. The pattern of RNA editing was highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites was confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites were enriched in GTEx RNA-sequencing data, edited ∼twentyfold higher than all other sites, and variation in RNA editing was predominantly explained by neuronal proportions in bulk brain tissue. Finally, we discovered 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. Lastly, we demonstrated barcoded calling cards can record binding in vivo in the mouse brain. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells.

Background: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Distributed “Point-of-Care” or “at-Home” testing is an important component for a complete suite of testing solutions. This manuscript describes the construction and operation of a platform technology designed to meet this need. The ongoing COVID-19 pandemic will be used as the proof-of-concept for the efficacy and deployment of this platform. The technology outlined consists of a one-pot, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) chemistry coupled with a low-cost and user-assembled reader using saliva as input. This platform is readily adapted to a wide range of pathogens due to the genetic basis of the reaction. A complete guide to the construction of the reader as well as the production of the reaction chemistry are provided here. Additionally, analytical limit of detection data and the results from saliva testing of SARS-CoV-2, are presented. The platform technology outlined here demonstrates a rapid, distributed, molecular point-of-care solution for pathogen detection using crude sample input.

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease–associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)–associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (ADNP, ARID1B, ASH1L, CHD2, and DYRK1A) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.

This protocol is a modification of Moudgil et al's Bulk Calling Cards Library Preparation that uses barcoded self-reporting transposons for Calling Cards and barcoded oligos for transcriptome capture. This protocol describes how to create next-generation sequencing libraries from barcoded self-reporting transposon (SRT) Calling Cards experiments to measure transcription factor binding and gene expression in parallel from collected RNA. This protocol assumes you have successfully transformed cells with barcoded piggyBac self-reporting transposons and either undirected piggyBac transposase or a gene expression regulator (GER) fused to piggyBac. Your cells are now ready for RNA extraction, SRT and transcriptome amplification, and library preparation.