Biomedical subjects

Brian C Thomas

Publications and source records attributed to Brian C Thomas.

3 recordsLinked to original sources

PubMed2025

Integration of therapeutic cargo into the human genome with programmable type V-K CAST.

CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.

Humans↗

PubMed2003

Gene structure and minimal promoter of mouse rdh1.

Mouse rdh1 encodes retinol dehydrogenase type 1 (RDH1), a short-chain dehydrogenase, which recognizes as substrates all-trans-retinol, 9-cis-retinol, 5alpha-androstan-3,17-diol and 5alpha-androstan-3-ol-17-one. RDH1 is the most efficient known mouse short-chain dehydrogenase that catalyzes dehydrogenation of all-trans-retinol, and contributes to a reconstituted path of all-trans-retinoic acid biosynthesis, when coexpressed in reporter cells with any one of three retinal dehydrogenases. Rdh1 shows widespread, if not ubiquitous, mRNA expression in the mouse beginning no later than embryo day 7. Here we report genomic organization, chromosomal localization and analysis of a minimum promoter of mouse rdh1. Rdh1 consists of four exons and three introns and spans approximately 14412 bp. Rdh1 is a single copy gene that maps to chromosome 10D3 with rdh5-9, but no known disorder maps precisely to rdh1. Rdh1 has three transcription start sites in kidney and one start site in liver. The rdh1 5'-region between -424 and +43 induces transcription maximally in COS7, mouse kidney RAG, and mouse liver NMu3Li cells. This section has no TATA box, but has a CCAAT box beginning 65 bp upstream of the major transcription start site, which is required for transcription of transfected reporter constructs. An AP1 binding site at -119 also activates transfected reporter constructs, and mediates 2-O-tetradecanoylphorbol-13-acetate (TPA) induced transcription. All-trans-retinoic acid antagonizes the TPA affect; however, no RARE or RXRE was found in the proximal promoter region, consistent with indirect regulation by all-trans-retinoic acid.

5' Flanking Region↗

PubMed2003

Conserved noncoding sequences in the grasses.

As orthologous genes from related species diverge over time, some sequences are conserved in noncoding regions. In mammals, large phylogenetic footprints, or conserved noncoding sequences (CNSs), are known to be common features of genes. Here we present the first large-scale analysis of plant genes for CNSs. We used maize and rice, maximally diverged members of the grass family of monocots. Using a local sequence alignment set to deliver only significant alignments, we found one or more CNSs in the noncoding regions of the majority of genes studied. Grass genes have dramatically fewer and much smaller CNSs than mammalian genes. Twenty-seven percent of grass gene comparisons revealed no CNSs. Genes functioning in upstream regulatory roles, such as transcription factors, are greatly enriched for CNSs relative to genes encoding enzymes or structural proteins. Further, we show that a CNS cluster in an intron of the knotted1 homeobox gene serves as a site of negative regulation. We showthat CNSs in the adh1 gene do not correlate with known cis-acting sites. We discuss the potential meanings of CNSs and their value as analytical tools and evolutionary characters. We advance the idea that many CNSs function to lock-in gene regulatory decisions.

5' Flanking Region↗