Brotman Baty Institute

ABSTRACT The human opsin gene cluster at Xq28 contains highly similar OPN1LW and OPN1MW genes essential for red-green color vision. Current molecular methods cannot accurately analyze this complex locus, limiting diagnosis of color vision deficiencies (CVD) and detection of carrier status. We performed Nanopore long-read sequencing of 206 individuals, comparing alignment-based analysis with targeted de novo assembly. Alignment-based methods performed poorly, whereas targeted assembly achieved 99% concordance for OPN1LW and 92% for OPN1MW copy numbers and resolved gene order in all XY individuals and 87% of XX individuals. This approach detected CVD in 3.2% of XY individuals and identified 8% of XX individuals as carriers, consistent with population estimates. Moreover, it molecularly explained the phenotypic severity in a family with Bornholm eye disease and clarified carrier status in an XX individual suspected of carrying two CVD haplotypes. Our approach provides a comprehensive, reference-free method for accurate analysis of expressed opsin genes and reliable CVD carrier detection.

Efficient genome writing in mammalian cells requires robust methods for integrating large DNA payloads. The previously described method mammalian Switching Antibiotic resistance markers Progressively for Integration (mSwAP-In) enables iterative, biallelic genome rewriting in mammalian stem cells with DNA payloads exceeding 100 kb. However, the lack of standardized vectors and certain technical constraints have limited its broader adoption. Here we present an improved plasmid toolkit designed to streamline the implementation of mSwAP-In. The toolkit includes two core vectors. pLP-TK (pCTC174) is a landing-pad plasmid compatible with Golden Gate assembly of genomic homology arms and supports both mSwAP-In and the recombinase-mediated cassette exchange method Big-IN. mSwAP-In MC2v2 (pKBA135) is a versatile Big DNA assembly and delivery vector that supports Gibson-based assembly and incorporates positive, negative, and fluorescent selection markers, as well as a backbone counterselection cassette to minimize unwanted plasmid integration. The vector architecture also enables propagation in yeast and bacterial hosts, inducible plasmid copy-number amplification in standard E. coli strains, and CRISPR/Cas9-mediated payload release through preinstalled guide RNA target sites. We further characterize the FCU1/5-FC counterselection system in mouse embryonic stem cells and define conditions that minimize its bystander toxicity. Finally, we provide a set of Cas9-gRNA expression plasmids optimized for common mSwAP-In applications. Together, these reagents constitute a standardized and experimentally validated toolkit that simplifies large-scale genome writing using mSwAP-In.

Molecular recording is an emerging paradigm for measuring biology over time. Enhancer-mediated genomic recording of activity in multiplex (ENGRAM) is a recently described synthetic biology circuit architecture that converts the transient activity of cis-regulatory elements (CREs) into stable genomic records that can be retrospectively recovered via DNA sequencing. Here we provide a step-by-step protocol for conducting ENGRAM experiments and analyzing the resulting data. We also describe key design considerations for ENGRAM recorders, summarize the strengths and limitations of ENGRAM, and highlight applications, including multiplex signal recording and high-throughput CRE screening. In contrast to other systems for DNA-based recording in mammalian systems, ENGRAM relies on prime editing-mediated insertions to record the activity of a given CRE, such that it is inherently multiplexable-for example, four-base-pair insertions can represent the activities of up to 256 distinct CREs. A further contrast lies with ENGRAM's compatibility with DNA Typewriter, which facilitates the capture of signal order. For users with basic skills in molecular biology, mammalian cell culture and DNA sequencing analysis, ENGRAM experiments can typically be completed within 5-6 weeks.

This collection details the saturation genome editing workflow.

BackgroundGermline pathogenic variants can inform targeted therapy for metastatic prostate cancer (mPC), and improve cancer early detection and risk reduction for family members. Guidelines recommend germline genetic testing be offered to all men with mPC, yet uptake of testing is only 10%–12%.MethodsThis prospective study enrolled veterans participating in the VA Million Veteran Program (MVP) with a diagnosis of mPC. Veterans were contacted by mail with option to opt‐out of future contact. Eligible veterans who did not opt‐out were mailed study information and received a follow‐up phone call to establish interest in germline testing. Participants provided verbal consent and were mailed a saliva collection kit for a CLIA‐level multigene cancer predisposition gene panel test. Results were disclosed to the patient and oncology provider. All steps were performed with genetic counseling support.ResultsOf 2104 eligible patients, 1952 veterans with mPC did not opt out. Of these, 681 (35%) provided consent and 459 (24%) completed testing. Of those who were approached 63% were White and 25% were Black. Fifty‐nine (13%) of those completing testing carried a germline pathogenic variant in a cancer risk gene. Of the 37 eligible for targeted therapy, 14 received targeted therapy, 18 did not yet have an indication for that therapy, and five were deceased without having received targeted therapy.ConclusionsParticipant completion of remote germline testing was facilitated at rates higher than the 10% previously reported. Remote genetic testing can augment uptake of testing in large, integrated health care systems.

The sense of smell has potent effects on appetite, but the underlying neural pathways remain undefined. Here, we investigated how olfactory signals reach two subsets of appetite-linked ("appetite") neurons in the hypothalamic arcuate nucleus: Agouti-related peptide (AgRP) neurons, which stimulate appetite, and POMC (pro-opiomelanocortin) neurons, which suppress it. Using polysynaptic viral tracing, we show that AgRP and POMC neurons receive indirect input from partially overlapping but distinct areas of the olfactory cortex, indicating that they process different sets of olfactory information. We also identify different complements of neurons more directly upstream of AgRP and POMC neurons that could relay olfactory cortical signals to the appetite neurons. Single-cell transcriptomics shows heterogeneous expression of neuromodulator receptors among AgRP neurons, suggesting variations in the signals they receive. Integrated viral tracing and RNA localization further reveals selected brain areas where upstream neurons express cognate receptor ligands. Together, these findings outline multiple pathways by which distinct olfactory and modulatory signals are differentially routed to neurons that promote versus inhibit appetite.

Variant interpretation remains one of the most significant challenges in clinical genetics. Variants of uncertain significance (VUS) undermine precision medicine implementation because they have an unknown relationship to disease and cannot be used for clinical decision-making. While evidence from multiplexed assays of variant effect (MAVEs) and other functional assays can help classify variants, major barriers prevent routine use in clinical variant classification, including fragmentation across multiple repositories, insufficient data standards, and the need to calibrate assays clinically. Here we address these challenges by presenting a new interface for the MaveDB database called MaveMD (MAVEs for MeDicine) that integrates with external resources such as ClinVar and the ClinGen Allele Registry, displays clinical evidence calibrations, provides intuitive visualizations, and exports structured evidence compatible with ACMG/AMP variant classification guidelines. MaveMD implements automatic mapping of MaveDB datasets to the human reference genome, dramatically simplifying the clinical translation of new MAVE data. We also defined a new metadata model after curating 438,318 variant effect measurements from 74 MAVE datasets spanning 32 disease-associated genes, and created an interface aimed at enabling effective clinical decision-making. Thus, MaveMD makes MAVE data accessible and easily usable for variant classification, and will scale seamlessly with future data generation efforts to empower the use of MAVE evidence in clinical practice.

Genetic risk prediction for Alzheimer's disease (AD) has high potential impact, yet few studies have assessed the reliability of various polygenic risk score (PRS) methods at the individual level. Here, we evaluated the reliability of AD PRS estimates among 6338 participants from the Multi-Ethnic Study of Atherosclerosis. We compared four PRS models that have been previously associated with dementia risk. Despite similar population-level performance metrics, inter-model reliability of individual-level risk assessment was low, even among individuals classified in the top and bottom deciles. These findings raise serious concerns about the downstream application of PRS for guiding interventions for AD.