Biology subjects

Mighell, T. L.

Publications and source records attributed to Mighell, T. L..

3 recordsLinked to original sources

bioRxiv2020

Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive GC sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogramed guide RNAs to direct CRISPR-Cas12a cleavage of double stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7,176 guides targeting 3.5 megabases of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 megabases. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for SNVs, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double stranded DNA template or genome.

genomics↗

bioRxiv2020

A sort-seq approach to the development of single fluorescent protein biosensors

The utility of single fluorescent protein biosensors (SFPBs) in biological research is offset by the difficulty in engineering these tools. SFPBs generally consist of three basic components: a circularly permuted fluorescent protein, a ligand-binding domain, and a pair of linkers connecting the two domains. In the absence of predictive methods for biosensor engineering, most designs combining these three components will fail to produce allosteric coupling between ligand binding and fluorescence emission. Methods to construct libraries of biosensor designs with variations in the site of GFP insertion and linker sequences have been developed, however, our ability to construct new variants has exceeded our ability to test them for function. Here, we address this challenge by applying a massively parallel assay termed "sort-seq" to the characterization of biosensor libraries. Sort-seq combines binned fluorescence-activated cell sorting, next-generation sequencing, and maximum likelihood estimation to quantify the dynamic range of many biosensor variants in parallel. We applied this method to two common biosensor optimization tasks: choice of insertion site and optimization of linker sequences. The sort-seq assay applied to a maltose-binding protein domain-insertion library not only identified previously described high-dynamic-range variants but also discovered new functional insertion-sites with diverse properties. A sort-seq assay performed on a pyruvate biosensor linker library expressed in mammalian cell culture identified linker variants with substantially improved dynamic range. Machine learning models trained on the resulting data can predict dynamic range from linker sequence. This high-throughput approach will accelerate the design and optimization of SFPBs, expanding the biosensor toolbox.

biochemistry↗

bioRxiv2018

A saturation mutagenesis approach to understanding PTEN lipid phosphatase activity and genotype-phenotypes relationships

Phosphatase and tensin homolog (PTEN) is a tumor suppressor frequently mutated in diverse cancers. Germline PTEN mutations are also associated with a range of clinical outcomes, including PTEN hamartoma tumor syndrome (PHTS) and autism spectrum disorder (ASD). To empower new insights into PTEN function and clinically relevant genotype-phenotype relationships, we systematically evaluated the effect of PTEN mutations on lipid phosphatase activity in vivo. Using a massively parallel approach that leverages an artificial humanized yeast model, we derived high-confidence estimates of functional impact for 7,244 single amino acid PTEN variants (86% of possible). These data uncovered novel insights into PTEN protein structure, biochemistry, and mutation tolerance. Variant functional scores can reliably discriminate likely pathogenic from benign alleles. Further, 32% of ClinVar unclassified missense variants are phosphatase deficient in our assay, supporting their reclassification. ASD associated mutations generally had less severe fitness scores relative to PHTS associated mutations (p = 7.16x10-5) and a higher fraction of hypomorphic mutations, arguing for continued genotype-phenotype studies in larger clinical datasets that can further leverage these rich functional data.

genomics↗