Rapid progress in the development of next-generation sequencing (NGS) technologies in recent years has provided many valuable insights into complex biological systems, ranging from cancer genomics to diverse microbial communities. NGS-based technologies for genomics, transcriptomics, and epigenomics are now increasingly focused on the characterization of individual cells. These single-cell analyses will allow researchers to uncover new and potentially unexpected biological discoveries relative to traditional profiling methods that assess bulk populations. Single-cell RNA sequencing (scRNA-seq), for example, can reveal complex and rare cell populations, uncover regulatory relationships between genes, and track the trajectories of distinct cell lineages in development. In this review, we will focus on technical challenges in single-cell isolation and library preparation and on computational analysis pipelines available for analyzing scRNA-seq data. Further technical improvements at the level of molecular and cell biology and in available bioinformatics tools will greatly facilitate both the basic science and medical applications of these sequencing technologies.

The authors developed a method for analyzing single cells under various conditions by tagging cells with small fragments of DNA.

DNA methylation is an epigenetic mechanism that is related to mammalian cellular differentiation, gene expression regulation, and disease. In several studies, DNA methylation has been identified as an effective marker to identify differences between cells. In this review, we introduce single-cell DNA-methylation profiling methods, including experimental strategies and approaches to computational data analysis. Furthermore, the blind spots of the basic analysis and recent alternatives are briefly described. In addition, we introduce well-known applications and discuss future development.

Mutations within the KRAS oncogene are associated with the proliferation of various cancers. Therapeutic approaches for treating cancers with such mutations have focused on targeting the downstream protein effectors of KRAS. However, to date, no approved treatment has targeted the mutated KRAS oncogene directly. Presently, we used the selectivity of the CRISPR/Cas9 system to directly target mutated KRAS alleles. We designed single-guide RNAs (sgRNAs) to target two specific single-nucleotide missense mutations on KRAS codon-12 located in the seed region adjacent to a protospacer adjacent motif (PAM). Lentiviral transduction of Cas9 and the sgRNAs into cancer cells with respective KRAS mutations resulted in high frequency of indels in the seed region. Indel-associated disruption of the mutant KRAS alleles correlated with reduced viability of the cancer cells. The results indicate that CRISPR-Cas9-mediated genome editing can potentially be used for the treatment of cancer patients, specifically those with oncogenic KRAS mutations.