Biology subjects

Reimer, K. A.

Publications and source records attributed to Reimer, K. A..

3 recordsLinked to original sources

bioRxiv2026

Engineered Allosteric Control Enhances Specificity and Potency of CRISPR-CasX-based Epigenetic Repressors

Epigenetic silencers are targeted therapeutics capable of persistently silencing genes without altering DNA sequence. However, many current silencers rely on direct fusion of a constitutively active DNA methyltransferase catalytic domain, that may cause off-target DNA methylation and cellular toxicity. In contrast, endogenous DNA methyltransferases contain a regulatory domain (ADD) that requires recognition of a permissive histone state before DNA methylation can occur. We reinstate this sequential proofreading logic in a compact CasX-based epigenetic repressor, generating Epigenetic Long-term CasX Repressors (ELXRs) that read local chromatin state before writing DNA methylation. In ELXRs, a repressor domain converts active chromatin into a state that unlocks the allosteric DNA methyltransferase. This creates a sequential, multi-gated process that preferentially restricts DNA methylation to the target site. Surprisingly, implementing this proofreading mechanism improved not only specificity, but also activity. ELXRs decreased off-target methylation up to 10-fold and rescued DNMT3A-dependent growth defects, with transcriptome profiling showing up to 150-fold fewer dysregulated genes, while also increasing on-target repression up to 4-fold across multiple loci. In mouse models, lipid nanoparticle delivery produced potent, durable PCSK9 silencing with precise promoter methylation. Together, these results demonstrate that coupling molecular recognition to effector activity through sequential proofreading can simultaneously improve specificity and potency.

molecular biology↗

bioRxiv2025

Long-read Sequencing of Nascent RNA from Budding and Fission Yeasts

Gene expression requires DNA transcription and simultaneous RNA processing steps that transform the precursor RNA into fully mature RNA. In eukaryotes, the processing of protein-encoding messenger RNAs (mRNAs) includes 5 end capping, editing, splicing, RNA modification, poly-adenylation cleavage, and polyadenylation. Short-read sequencing of total or messenger RNA largely reveals the final output of transcription and processing because it utilizes 1) steady-state, mature RNA that is mostly processed and 2) sequencing reads that are too short to detect adjacent processing events (e.g. two adjacent introns). In contrast, long-read sequencing of nascent RNA allows the detection of rarer, full-length transcripts that are in the process of being transcribed and processed. The 3 end of each nascent RNA establishes the position of RNA polymerase II (Pol II) along the gene at the time of cell lysis, providing a timeline for RNA processing events. In addition, the density of 3 ends along genes or at gene landmarks reflects Pol II density, which is related to changes in transcription elongation rate. In organisms with complex gene architectures, information about splicing across multiple introns within the same transcript can be extracted, as well as the location of transcription start sites (TSSs) and polyA cleavage sites. Here, we describe the isolation of nascent RNA from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, preparation of a cDNA library for long-read sequencing on Oxford Nanopore Technologies or Pacific Biosciences platforms, and initial data analysis steps. These methods comprise versatile and powerful tools for the investigation of coupled RNA synthesis and processing.

molecular biology↗

bioRxiv2020

Rapid and Efficient Co-Transcriptional Splicing Enhances Mammalian Gene Expression

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3 end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and PRO-seq during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75 - 300 nucleotides of 3 splice sites (3SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3 end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3SS improves both splicing and 3 end cleavage of individual {beta}-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.

molecular biology↗