bims-dresag 2021-08-08 papers

Issue of 2021–08–08
four papers selected by
Pengyi Yan, Shanghai Jiao Tong University

Nat Protoc. 2021 Aug 06.

Identification of cancer-related mutations in human pluripotent stem cells using RNA-seq analysis.

Human pluripotent stem cells (hPSCs) are known to acquire genetic aberrations during in vitro propagation. In addition to recurrent chromosomal aberrations, it has recently been shown that these cells also gain point mutations in cancer-related genes, predominantly in TP53. The need for routine quality control of hPSCs is critical for both basic research and clinical applications. Here we discuss the relevance of detecting mutations for various hPSCs applications, and present a detailed protocol to identify cancer-related point mutations using data from RNA sequencing, an assay commonly performed during the growth and differentiation of hPSCs. In this protocol, we describe how to process and align the sequencing data, analyze it and conservatively interpret the results in order to generate an accurate estimation of mutations in tumor-related genes. This pipeline is designed to work in high throughput and is available as a software container at https://github.com/elyadlezmi/RNA2CM . The protocol requires minimal command-line skills and can be carried out in 1-2 d.

DOI: https://doi.org/10.1038/s41596-021-00591-5

Mol Cell. 2021 Aug 05. pii: S1097-2765(21)00575-X. [Epub ahead of print]81(15): 3046-3047

BARBEKO'ing in the lab: Versatile CRISPR screens with barcoded base editors.

Pranam Chatterjee.

Xu et al. (2021) describe a novel two-pronged CRISPR screen, termed BARBEKO, by coupling cytosine base editors and internally barcoded sgRNAs to eliminate double-stranded break-induced toxicity, enable high multiplicities of infection, and ensure experimental reproducibility.

DOI: https://doi.org/10.1016/j.molcel.2021.07.007

Nat Methods. 2021 Aug 05.

Augmenting and directing long-range CRISPR-mediated activation in human cells.

Y Esther Tak, Joy E Horng, Nicholas T Perry, Hayley T Schultz, Sowmya Iyer, Qiuming Yao, Luli S Zou, Martin J Aryee, Luca Pinello, J Keith Joung.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

DOI: https://doi.org/10.1038/s41592-021-01224-1

Elife. 2021 Aug 03. pii: e59828. [Epub ahead of print]10

Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.

Knut H Lauritzen, Maria Belland Olsen, Mohammed Shakil Ahmed, Kuan Yang, Johanne Egge Rinholm, Linda H Bergersen, Qin Ying Esbensen, Lars Jansen Sverkeli, Mathias Ziegler, Håvard Attramadal, Bente Halvorsen, Pål Aukrust, Arne Yndestad.

Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.

Keywords: DNA repair; NAD+; SIRT3; biochemistry; cardiovascular disease; chemical biology; human; mitochondrial dna; mouse; nicotinamide riboside

DOI: https://doi.org/10.7554/eLife.59828