The induced pluripotent stem cell (iPSC) technology has transformed in vitro research and holds great promise to advance regenerative medicine. iPSCs have the capacity for an almost unlimited expansion, are amenable to genetic engineering, and can be differentiated into most somatic cell types. iPSCs have been widely applied to model human development and diseases, perform drug screening, and develop cell therapies. In this review, we outline key developments in the iPSC field and highlight the immense versatility of the iPSC technology for in vitro modeling and therapeutic applications. We begin by discussing the pivotal discoveries that revealed the potential of a somatic cell nucleus for reprogramming and led to successful generation of iPSCs. We consider the molecular mechanisms and dynamics of somatic cell reprogramming as well as the numerous methods available to induce pluripotency. Subsequently, we discuss various iPSC-based cellular models, from mono-cultures of a single cell type to complex three-dimensional organoids, and how these models can be applied to elucidate the mechanisms of human development and diseases. We use examples of neurological disorders, coronavirus disease 2019 (COVID-19), and cancer to highlight the diversity of disease-specific phenotypes that can be modeled using iPSC-derived cells. We also consider how iPSC-derived cellular models can be used in high-throughput drug screening and drug toxicity studies. Finally, we discuss the process of developing autologous and allogeneic iPSC-based cell therapies and their potential to alleviate human diseases. 

Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications

The clustered regularly interspaced short palindromic repeats (CRISPR)‐based genetic screening has been demonstrated as a powerful approach for unbiased functional genomics research. Single‐cell CRISPR screening (scCRISPR) techniques, which result from the combination of single‐cell toolkits and CRISPR screening, allow dissecting regulatory networks in complex biological systems at unprecedented resolution. These methods allow cells to be perturbed en masse using a pooled CRISPR library, followed by high‐content phenotyping. This is technically accomplished by annotating cells with sgRNA‐specific barcodes or directly detectable sgRNAs. According to the integration of distinct single‐cell technologies, these methods principally fall into four categories: scCRISPR with RNA‐seq, scCRISPR with ATAC‐seq, scCRISPR with proteome probing, and imaging‐based scCRISPR. scCRISPR has deciphered genotype–phenotype relationships, genetic regulations, tumor biological issues, and neuropathological mechanisms. This review provides insight into the technical breakthrough of scCRISPR by systematically summarizing the advancements of various scCRISPR methodologies and analyzing their merits and limitations. In addition, an application‐oriented approach guide is offered to meet researchers’ individualized demands.

Massively Parallel CRISPR‐Based Genetic Perturbation Screening at Single‐Cell Resolution

Base editor screens for in situ mutational scanning at scale.

Single-cell sequencing is a powerful approach that can detect genetic alterations and their phenotypic consequences in the context of human development, with cellular resolution. Humans start out as single-cell zygotes and undergo fission and differentiation to develop into multicellular organisms. Before fertilisation and during development, the cellular genome acquires hundreds of mutations that propagate down the cell lineage. Whether germline or somatic in nature, some of these mutations may have significant genotypic impact and lead to diseased cellular phenotypes, either systemically or confined to a tissue. Single-cell sequencing enables the detection and monitoring of the genotype and the consequent molecular phenotypes at a cellular resolution. It offers powerful tools to compare the cellular lineage between ‘normal’ and ‘diseased’ conditions and to establish genotype-phenotype relationships. By preserving cellular heterogeneity, single-cell sequencing, unlike bulk-sequencing, allows the detection of even small, diseased subpopulations of cells within an otherwise normal tissue. Indeed, the characterisation of biopsies with cellular resolution can provide a mechanistic view of the disease. While single-cell approaches are currently used mainly in basic research, it can be expected that applications of these technologies in the clinic may aid the detection, diagnosis and eventually the treatment of rare genetic diseases as well as cancer. This review article provides an overview of the single-cell sequencing technologies in the context of human genetics, with an aim to empower clinicians to understand and interpret the single-cell sequencing data and analyses. We discuss the state-of-the-art experimental and analytical workflows and highlight current challenges/limitations. Notably, we focus on two prospective applications of the technology in human genetics, namely the annotation of the non-coding genome using single-cell functional genomics and the use of single-cell sequencing data for in silico variant prioritisation. 

/pdf/the-role-of-single-cell-genomics-in-human-genetics-1ikfa5w2.pdf

The role of single-cell genomics in human genetics

Abstract Background While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N 6 -methyladenosine (m 6 A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. Methods Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. Results METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a ( eIF3a ) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). Conclusions Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency. 

METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation

Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present “sc-Tiling,” which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response. Identifying functional domains and genetic regulatory mechanisms is essential for developing new therapies. Here the authors present sc-Tiling, single-cell high-density CRISPR tiling screening for functional domain characterization.

/pdf/high-resolution-characterization-of-gene-function-using-4ollw83d76.pdf

High-resolution characterization of gene function using single-cell CRISPR tiling screen.

Sphingolipids and their metabolic pathways have been implicated in disease development and therapeutic response; however, the detailed mechanisms remain unclear. Using a sphingolipid network focused CRISPR/Cas9 library screen, we identified an endoplasmic reticulum (ER) enzyme, 3-Ketodihydrosphingosine reductase (KDSR), to be essential for leukemia cell maintenance. Loss of KDSR led to apoptosis, cell cycle arrest, and aberrant ER structure. Transcriptomic analysis revealed the indispensable role of KDSR in maintaining the unfolded protein response (UPR) in ER. High-density CRISPR tiling scan and sphingolipid mass spectrometry pinpointed the critical role of KDSR's catalytic function in leukemia. Mechanistically, depletion of KDSR resulted in accumulated 3-ketodihydrosphingosine (KDS) and dysregulated UPR checkpoint proteins PERK, ATF6, and ATF4. Finally, our study revealed the synergism between KDSR suppression and pharmacologically induced ER-stress, underscoring a therapeutic potential of combinatorial targeting sphingolipid metabolism and ER homeostasis in leukemia treatment.

3-Ketodihydrosphingosine reductase maintains ER homeostasis and unfolded protein response in leukemia.

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High-resolution characterization of gene function using single-cell CRISPR tiling screen.

3-Ketodihydrosphingosine reductase maintains ER homeostasis and unfolded protein response in leukemia.