Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations.

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape.

CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9-binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

TERRA, hnRNP A1, and DNA-PKcs Interactions at Human Telomeres.

Maintenance of telomeres, repetitive elements at eukaryotic chromosomal termini, and the end-capping structure and function they provide, are imperative for preserving genome integrity and stability. The discovery that telomeres are transcribed into telomere repeat containing RNA (TERRA) has revolutionized our view of this repetitive, rather unappreciated region of the genome. We have previously shown that the non-homologous end-joining, shelterin associated DNA dependent protein kinase catalytic subunit (DNA-PKcs) participates in mammalian telomeric end-capping, exclusively at telomeres created by leading-strand synthesis. Here, we explore potential roles of DNA-PKcs and its phosphorylation target heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in the localization of TERRA at human telomeres. Evaluation of co-localized foci utilizing RNA-FISH and three-dimensional (3D) reconstruction strategies provided evidence that both inhibition of DNA-PKcs kinase activity and siRNA depletion of hnRNP A1 result in accumulation of TERRA at individual telomeres; depletion of hnRNP A1 also resulted in increased frequencies of fragile telomeres. These observations are consistent with previous demonstrations that decreased levels of the nonsense RNA-mediated decay factors SMG1 and UPF1 increase TERRA at telomeres and interfere with replication of leading-strand telomeres. We propose that hTR mediated stimulation of DNA-PKcs and subsequent phosphorylation of hnRNP A1 influences the cell cycle dependent distribution of TERRA at telomeres by contributing to the removal of TERRA from telomeres, an action important for progression of S-phase, and thereby facilitating efficient telomere replication and end-capping.