Biology subjects

Waters, A. J.

Publications and source records attributed to Waters, A. J..

5 recordsLinked to original sources

bioRxiv2026

SELECT-seq allows Pre-Sequencing Enrichment of SNP Edits in One-Pot Single-Cell Whole-Transcriptome Sequencing

Advances in high-throughput sequencing have associated millions of putative genetic variants with disease. However, scalable experimental methods to establish causal relationships between genetic variants and downstream transcriptional outcomes remain a major challenge. Single-cell methods that integrate genotyping with transcriptomic profiling provide a way to address this, but do not enable pre-sequencing enrichment of correctly edited cells, limiting scale. We present SELECT-seq (SNP Enrichment Leveraging Cas12a Targeting), a rapid method that allows SNP-specific PCR amplification and Cas12a-mediated fluorescence detection simultaneously with whole-transcriptome amplification. This one-pot workflow enables identification and enrichment of SNP-bearing single cells, making a rapid and scalable methodology for analysis of genotype-phenotype linkage avoiding laborious single cell cloning steps. As a proof of principle we show that SELECT-seq distinguishes U-2 OS and T-47D cell lines based on a PIK3CA (NM_006218.4:c.3463A>G) mutation while preserving transcriptome integrity. It physically enriches a rare NRF2 T80K (NM_006164.5:c.390C>A) mutant cells (6.7%) from a prime-edited pool, achieving 86% genotype accuracy, and shows 87.5% directional concordance in the transcriptomic effects compared with a clonal NRF2 T80K cell line. SELECT-seq thus provides a rapid, scalable and widely accessible approach for mapping genotype-phenotype relationships at single-cell resolution.

molecular biology↗

bioRxiv2026

Saturation-seq integrates single-cell saturation genome editing and RNA-seq to quantify NFE2L2 (NRF2) variant effects

Interpreting the functional consequences of variants remains one of the central unsolved problems in genomics and clinical genetics. Compounding this, most existing approaches rely on reductive, one-dimensional proxies such as cell growth to score variant effects, which can be a poor substitute for the rich, multidimensional phenotyping that is ultimately needed to understand how variants alter biology. This is especially true for variants known to act through gain-of-function/neomorphic mechanisms. We developed Saturation-seq, a high-throughput platform that combines saturation genome editing with single-cell DNA and RNA profiling to systematically map variant effects. Using CRISPR-based editing in a barcoded haploid cell line, we install hundreds of variants directly into endogenous genomic loci, testing them in multiplex and preserving the native coding and regulatory context. Single-cell amplicon and transcriptome sequencing enables direct linkage of each genomic edit to its transcriptional impact. We apply Saturation-seq to comprehensively characterize 230 variants in the recurrently mutated N-terminal region of NFE2L2 (NRF2), a master regulator of oxidative stress and an oncogene mutated in lung cancer. We define variant function with disruption scores computed from misregulation of known NRF2 targets in single-cell transcriptomes; scores separate pathogenic/benign truthset variants with >90% accuracy and enabled interpretation of TCGA and TRACERx patient tumor data, as well as a rare NFE2L2 germline variant linked to a developmental syndrome. Thus, we establish a broadly applicable high-resolution single-cell variant-to-function platform with a rich phenotypic readout.

genomics↗

bioRxiv2025

Comprehensively Testing the Function of Missense Variation in the STK11 Tumour Suppressor

The tumor suppressor gene STK11 encoding Serine/Threonine Kinase 11 (STK11) is associated with Peutz-Jeghers Syndrome (PJS), a heritable gastrointestinal disease that increases lifetime cancer risk, and with somatic variation that contributes to [~]30% of lung and 20% of cervical cancers. Although identifying pathogenic variants is clinically actionable, over 94% of STK11 missense variants that have been observed clinically lack a definitive classification. We therefore measured the impact of STK11 variants at scale in a mammalian cell-based assay, scoring 6,026 (73% of all possible) amino acid substitutions across the full-length gene. Functional scores--which were consistent with biochemical properties, smaller-scale assays, and pathogenicity annotations--identified a subset of PJS patients with germline STK11 variants diagnosed later in life, as well as somatic STK11 variants found in cancer patients that had comparable overall survival estimates to wild-type STK11. Our scores provided new evidence for 350 annotated VUS STK11 missense variants and [~]80% of missense variants that have not yet been reported clinically, but we might expect to observe in the future. Thus, our effect map provides a proactive resource for gaining sequence-structure-function insights and evidence for actionable interpretation of clinical missense variants.

cancer biology↗

bioRxiv2021

Variant Library Annotation Tool (VaLiAnT): an oligonucleotide library design and annotation tool for Saturation Genome Editing and other Deep Mutational Scanning experiments

MotivationRecent advances in CRISPR/Cas9 technology allow for the functional analysis of genetic variants at single nucleotide resolution whilst maintaining genomic context (Findlay et al., 2018). This approach, known as saturation genome editing (SGE), is a distinct type of deep mutational scanning (DMS) that systematically alters each position in a target region to explore its function. SGE experiments require the design and synthesis of oligonucleotide variant libraries which are introduced into the genome by homology-directed repair (HDR). This technology is broadly applicable to diverse research fields such as disease variant identification, drug development, structure-function studies, synthetic biology, evolutionary genetics and the study of host-pathogen interactions. Here we present the Variant Library Annotation Tool (VaLiAnT) which can be used to generate saturation mutagenesis oligonucleotide libraries from user-defined genomic coordinates and standardised input files. This software package is intentionally versatile to accommodate diverse operability, with species, genomic reference sequences and transcriptomic annotations specified by the user. Genomic ranges, directionality and frame information are considered to allow perturbations at both the nucleotide and amino acid level. ResultsCoordinates for a genomic range, that may include exonic and/or intronic sequence, are provided by the user in order to retrieve a corresponding oligonucleotide reference sequence. A user-specified range within this sequence is then subject to systematic, nucleotide and/or amino acid saturating mutator functions, with each discrete mutation returned to the user as a separate sequence, building up the final oligo library. If desired, variant accessions from genetic information repositories, such as ClinVar and gnomAD, that fall within the user-specified ranges, will also be incorporated into the library. For SGE library generation, base reference sequences can be modified to include PAM (Protospacer Adjacent Motif) and protospacer protection edits that prevent Cas9 from cutting incorporated oligonucleotide tracts. Mutator functions modify this protected reference sequence to generate variant sequences. Constant regions are designated for non-editing to allow specific adapter annealing for downstream cloning and amplification from the library pool. A metadata file is generated, delineating annotation information for each variant sequence to aid computational analysis. In addition, a library file is generated, which contains unique sequences (any exact duplicate sequences are removed) ready for submission to commercial synthesis platforms. A VCF file listing all variants is also generated for analysis and quality control processes. The VaLiAnT software package provides a novel means to systemically retrieve, mutate and annotate genomic sequences for oligonucleotide library generation. Specific features for SGE library generation can be employed, with other diverse applications possible. Availability and ImplementationVaLiAnT is a command line tool written in Python. Source code, testing data, example library input and output files, and executables are available at https://github.com/cancerit/VaLiAnT. A user manual details step by step instructions for software use, available at https://github.com/cancerit/VaLiAnT/wiki. The software is freely available for non-commercial use (see Licence for more details, https://github.com/cancerit/VaLiAnT/blob/develop/LICENSE).

genomics↗

bioRxiv2018

Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a miss-activation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

synthetic biology↗