Nipun Verma

An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) offer a unique platform for elucidating the genes and molecular pathways that underlie complex traits and diseases. To realize this promise, methods for rapid and controllable genetic manipulations are urgently needed. By combining two newly developed gene-editing tools, the TALEN and CRISPR/Cas systems, we have developed a genome-engineering platform in hPSCs, which we named iCRISPR. iCRISPR enabled rapid and highly efficient generation of biallelic knockout hPSCs for loss-of-function studies, as well as homozygous knockin hPSCs with specific nucleotide alterations for precise modeling of disease conditions. We further demonstrate efficient one-step generation of double- and triple-gene knockout hPSC lines, as well as stage-specific inducible gene knockout during hPSC differentiation. Thus the iCRISPR platform is uniquely suited for dissection of complex genetic interactions and pleiotropic gene functions in human disease studies and has the potential to support high-throughput genetic analysis in hPSCs.

A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells

Summary The development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.

CRISPR/Cas9-Based Engineering of the Epigenome

Determining causal relationships between distinct chromatin features and gene expression, and ultimately cell behavior, remains a major challenge. Recent developments in targetable epigenome-editing tools enable us to assign direct transcriptional and functional consequences to locus-specific chromatin modifications. This Protocol Review discusses the unprecedented opportunity that CRISPR/Cas9 technology offers for investigating and manipulating the epigenome to facilitate further understanding of stem cell biology and engineering of stem cells for therapeutic applications. We also provide technical considerations for standardization and further improvement of the CRISPR/Cas9-based tools to engineer the epigenome.

Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development

Human disease phenotypes associated with haploinsufficient gene requirements are often not recapitulated well in animal models. Here, we have investigated the association between human GATA6 haploinsufficiency and a wide range of clinical phenotypes that include neonatal and adult-onset diabetes using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome editing coupled with human pluripotent stem cell (hPSC) directed differentiation. We found that loss of one GATA6 allele specifically affects the differentiation of human pancreatic progenitors from the early PDX1+ stage to the more mature PDX1+NKX6.1+ stage, leading to impaired formation of glucose-responsive β-like cells. In addition to this GATA6 haploinsufficiency, we also identified dosage-sensitive requirements for GATA6 and GATA4 in the formation of both definitive endoderm and pancreatic progenitor cells. Our work expands the application of hPSCs from studying the impact of individual gene loci to investigation of multigenic human traits, and it establishes an approach for identifying genetic modifiers of human disease.

Induced pluripotent stem cells and promises of neuroregenerative medicine.

First created in 2006 from adult somatic cells by a simple molecular genetic trick, induced pluripotent stem cells (iPS) system is the latest platform in stem cell research. Induced pluripotent stem cells are produced by nuclear reprogramming technology and they resemble embryonic stem cells (ES) in key elements; they possess the potentiality to differentiate into any type of cell in the body. More importantly, the iPS platform has distinct advantage over ES system in the sense that iPS-derived cells are autologous and therefore the iPS-derived transplantation does not require immunosuppressive therapy. In addition, iPS research obviates the political and ethical quandary associated with embryo destruction and ES research. This remarkable discovery of cellular plasticity has important medical implications. This brief review summarizes currently available stem cell platforms, with emphasis on cellular reprogramming and iPS technology and its application in disease modeling and cell replacement therapy in neurodegenerative diseases.

Sildenafil citrate improves erectile function after castration in a rat model

The administration of phosphodiesterase 5 inhibitor commencing at the time of castration might preserve erectile function.

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP–seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit bivalent promoter hypermethylation without a corresponding decrease in gene expression in the undifferentiated state. However, PAX6 promoter hypermethylation in TKO hESCs impairs neural differentiation.

CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells

Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.

DICER1 Is Essential for Self-Renewal of Human Embryonic Stem Cells

Summary MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos.

Publisher Correction: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells

The version of the Supplementary Text and Figures file initially posted was missing Supplementary Tables 1–6 and the Supplementary Note and used incorrect versions of the supplementary figures.