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1. Accurate integration of single-cell DNA and RNA for analyzing intratumor heterogeneity using MaCroDNA

   https://doi.org/10.1038/s41467-023-44014-3  

   Edrisi, Mohammadamin; Huang, Xiru; Ogilvie, Huw A.; Nakhleh, Luay (December 2023, Nature Communications)

   Abstract Cancers develop and progress as mutations accumulate, and with the advent of single-cell DNA and RNA sequencing, researchers can observe these mutations and their transcriptomic effects and predict proteomic changes with remarkable temporal and spatial precision. However, to connect genomic mutations with their transcriptomic and proteomic consequences, cells with either only DNA data or only RNA data must be mapped to a common domain. For this purpose, we present MaCroDNA, a method that uses maximum weighted bipartite matching of per-gene read counts from single-cell DNA and RNA-seq data. Using ground truth information from colorectal cancer data, we demonstrate the advantage of MaCroDNA over existing methods in accuracy and speed. Exemplifying the utility of single-cell data integration in cancer research, we suggest, based on results derived using MaCroDNA, that genomic mutations of large effect size increasingly contribute to differential expression between cells as Barrett’s esophagus progresses to esophageal cancer, reaffirming the findings of the previous studies. 

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2. RNA-mediated double-strand break repair by end-joining mechanisms

   https://doi.org/10.1038/s41467-024-51457-9  

   Jeon, Youngkyu; Lu, Yilin; Ferrari, Margherita Maria; Channagiri, Tejasvi; Xu, Penghao; Meers, Chance; Zhang, Yiqi; Balachander, Sathya; Park, Vivian S; Marsili, Stefania; et al (December 2024, Nature Communications)

   Abstract Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution. 

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3. Gene Co-Expression Analysis of Human RNASEH2A Reveals Functional Networks Associated with DNA Replication, DNA Damage Response, and Cell Cycle Regulation

   https://doi.org/10.3390/biology10030221  

   Marsili, Stefania; Tichon, Ailone; Kundnani, Deepali; Storici, Francesca (March 2021, Biology)

   null (Ed.)

   Ribonuclease (RNase) H2 is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance, and DNA damage repair. RNase H2 is a trimer composed of three subunits, RNASEH2A being the catalytic subunit. RNASEH2A expression levels have been shown to be upregulated in transformed and cancer cells. In this study, we used a bioinformatics approach to identify RNASEH2A co-expressed genes in different human tissues to underscore biological processes associated with RNASEH2A expression. Our analysis shows functional networks for RNASEH2A involvement such as DNA replication and DNA damage response and a novel putative functional network of cell cycle regulation. Further bioinformatics investigation showed increased gene expression in different types of actively cycling cells and tissues, particularly in several cancers, supporting a biological role for RNASEH2A but not for the other two subunits of RNase H2 in cell proliferation. Mass spectrometry analysis of RNASEH2A-bound proteins identified players functioning in cell cycle regulation. Additional bioinformatic analysis showed that RNASEH2A correlates with cancer progression and cell cycle related genes in Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) Pan Cancer datasets and supported our mass spectrometry findings. 

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4. CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

   https://doi.org/10.1093/nar/gkac049  

   Lin-Shiao, Enrique; Pfeifer, Wolfgang G.; Shy, Brian R.; Saffari Doost, Mohammad; Chen, Evelyn; Vykunta, Vivasvan S.; Hamilton, Jennifer R.; Stahl, Elizabeth C.; Lopez, Diana M.; Sandoval Espinoza, Cindy R.; et al (February 2022, Nucleic Acids Research)

   Abstract DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei. 

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5. Biomaterial‐Based In Situ Cancer Vaccines

   https://doi.org/10.1002/adma.202210452  

   Bo, Yang; Wang, Hua (April 2023, Advanced Materials)

   Abstract Cancer immunotherapies have reshaped the paradigm for cancer treatment over the past decade. Among them, therapeutic cancer vaccines that aim to modulate antigen‐presenting cells and subsequent T cell priming processes are among the first FDA‐approved cancer immunotherapies. However, despite showing benign safety profiles and the capability to generate antigen‐specific humoral and cellular responses, cancer vaccines have been limited by the modest therapeutic efficacy, especially for immunologically cold solid tumors. One key challenge lies in the identification of tumor‐specific antigens, which involves a costly and lengthy process of tumor cell isolation, DNA/RNA extraction, sequencing, mutation analysis, epitope prediction, peptide synthesis, and antigen screening. To address these issues, in situ cancer vaccines have been actively pursued to generate endogenous antigens directly from tumors and utilize the generated tumor antigens to elicit potent cytotoxic T lymphocyte (CTL) response. Biomaterials‐based in situ cancer vaccines, in particular, have achieved significant progress by taking advantage of biomaterials that can synergize antigens and adjuvants, troubleshoot delivery issues, home, and manipulate immune cells in situ. This review will provide an overview of biomaterials‐based in situ cancer vaccines, either living or artificial materials, under development or in the clinic, and discuss the design criteria for in situ cancer vaccines. 

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