Biology subjects

McGee, A. V.

Publications and source records attributed to McGee, A. V..

6 recordsLinked to original sources

bioRxiv2026

Locus-Scale Massively Parallel Reporter Assays

Interactions among cis-regulatory elements (CREs) are central to mammalian gene regulation. Long @$$ massively parallel reporter assays (LAMPRAs) integrate combinatorial cloning, molecular barcoding and long- and short-read sequencing, to scalably measure how CRE identities, numbers, spacings, orders, orientations, and interactions shape regulatory output at multi-kilobase length scales. As a proof-of-concept, we assay 36,000 x 5-kb synthetic cis-regulatory loci (sCRLs), each a 5 x 1-kb random combination of enhancers, insulators and spacers, to model how locus composition drives gene expression.

genomics↗

bioRxiv2026

An integrated, scaled approach to resolve TSC2 variants of uncertain significance

Obtaining a precise genetic tuberous sclerosis diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance (VUS). VUS in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3,634 TSC2 missense VUS observed so far. To address this challenge, we used massively parallel sequencing to measure the steady-state abundance of almost 9,000 TSC2 missense variants and developed an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. 1,288 of 8,891 (14.49%) missense variants assayed had altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed had altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassified 212 of 276 (76.8%) TSC2 missense VUS. These datasets will lead to improved genetic diagnosis of tuberous sclerosis with potential positive impacts on the clinical management of patients and their families.

genetics↗

bioRxiv2023

Modular vector assembly enables rapid assessment of emerging CRISPR technologies

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

genomics↗

bioRxiv2023

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

genomics↗

bioRxiv2022

Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening

CRISPR technology is a powerful tool for studying genome function. To aid in picking sgRNAs that have maximal efficacy against a target of interest from many possible options, several groups have developed models that predict sgRNA on-target activity. Although multiple tracrRNA variants are commonly used for screening, no existing models account for this feature when nominating sgRNAs. Here we develop an on-target model, Rule Set 3, that makes optimal predictions for multiple tracrRNA variants. We validate Rule Set 3 on a new dataset of sgRNAs tiling essential and non-essential genes, demonstrating substantial improvement over prior prediction models. By analyzing the differences in sgRNA activity between tracrRNA variants, we show that Pol III transcription termination is a strong determinant of sgRNA activity. We expect these results to improve the performance of CRISPR screening and inform future research on tracrRNA engineering and sgRNA modeling.

bioengineering↗

bioRxiv2021

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we benchmark PAM preferences, on-target activity, and off-target susceptibility of 11 variants of SpCas9 in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A>G and C>T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new insights into these clinically-relevant genes. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

genomics↗