A robust and inducible precise genome editing via an all-in-one prime editor in human pluripotent stem cells.

Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small deletions, or insertions at a specific genomic locus, a strategy that shows great promise for creating "Disease in a dish" models. To improve the effectiveness of prime editing in hPSCs, we systematically compared and combined the "inhibition of mismatch repair pathway and p53" on top of the "PEmax" to generate an all-in-one "PE-Plus" prime editor. We show that PE-Plus conducts the most efficient editing among the current PE tools in hPSCs. We further established an inducible prime editing platform in hPSCs by incorporating the all-in-one PE vector into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Therefore, our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.

An integrative single-cell multi-omics profiling of human pancreatic islets identifies T1D associated genes and regulatory signals.

Genome wide association studies (GWAS) have identified over 100 signals associated with type 1 diabetes (T1D). However, translating any given T1D GWAS signal into mechanistic insights, including putative causal variants and the context (cell type and cell state) in which they function, has been limited. Here, we present a comprehensive multi-omic integrative analysis of single-cell/nucleus resolution profiles of gene expression and chromatin accessibility in healthy and autoantibody+ (AAB+) human islets, as well as islets under multiple T1D stimulatory conditions. We broadly nominate effector cell types for all T1D GWAS signals. We further nominated higher-resolution contexts, including effector cell types, regulatory elements, and genes for three independent T1D risk variants acting through islet cells within the pancreas at the DLK1/MEG3, RASGRP1, and TOX loci. Subsequently, we created isogenic gene knockouts DLK1-/-, RASGRP1-/-, and TOX-/-, and the corresponding regulatory region knockout, RASGRP1Δ, and DLK1Δ hESCs. Loss of RASGRP1 or DLK1, as well as knockout of the regulatory region of RASGRP1 or DLK1, increased ß cell apoptosis. Additionally, pancreatic ß cells derived from isogenic hESCs carrying the risk allele of rs3783355A/A exhibited increased ß cell death. Finally, RNA-seq and ATAC-seq identified five genes upregulated in both RASGRP1-/- and DLK1-/- ß-like cells, four of which are associated with T1D. Together, this work reports an integrative approach for combining single cell multi-omics, GWAS, and isogenic hESC-derived ß-like cells to prioritize the T1D associated signals and their underlying context-specific cell types, genes, SNPs, and regulatory elements, to illuminate biological functions and molecular mechanisms.

Functional interrogation of twenty type 2 diabetes-associated genes using isogenic human embryonic stem cell-derived ß-like cells.

Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional roles of many loci remain unexplored. Here, we engineered isogenic knockout human embryonic stem cell lines for 20 genes associated with T2D risk. We examined the impacts of each knockout on ß cell differentiation, functions, and survival. We generated gene expression and chromatin accessibility profiles on ß cells derived from each knockout line. Analyses of T2D-association signals overlapping HNF4A-dependent ATAC peaks identified a likely causal variant at the FAIM2 T2D-association signal. Additionally, the integrative association analyses identified four genes (CP, RNASE1, PCSK1N, and GSTA2) associated with insulin production, and two genes (TAGLN3 and DHRS2) associated with ß cell sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental line and identified a single likely functional variant at each of 23 T2D-association signals.

Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells.

Prime editing introduces single-nucleotide polymorphism changes, small deletions, or insertions at a specific genome site without double-stranded DNA breaks or the need for the donor template. Here, we present a protocol to design, conduct, and evaluate prime editing in human pluripotent stem cells. We describe steps for pegRNA and nicking sgRNA design and cloning, the prime editing tool electroporation, and the efficiency evaluation using Miseq. We elaborate the process of GBA (N370S) mutation induction and correction as an example. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1.

Isogenic human trophectoderm cells demonstrate the role of NDUFA4 and associated variants in ZIKV infection.

Population-based genome-wide association studies (GWAS) normally require a large sample size, which can be labor intensive and costly. Recently, we reported a human induced pluripotent stem cell (hiPSC) array-based GWAS method, identifying NDUFA4 as a host factor for Zika virus (ZIKV) infection. In this study, we extended our analysis to trophectoderm cells, which constitute one of the major routes of mother-to-fetus transmission of ZIKV during pregnancy. We differentiated hiPSCs from various donors into trophectoderm cells. We then infected cells carrying loss of function mutations in NDUFA4, harboring risk versus non-risk alleles of SNPs (rs917172 and rs12386620) or having deletions in the NDUFA4 cis-regulatory region with ZIKV. We found that loss/reduction of NDUFA4 suppressed ZIKV infection in trophectoderm cells. This study validated our published hiPSC array-based system as a useful platform for GWAS and confirmed the role of NDUFA4 as a susceptibility locus for ZIKV in disease-relevant trophectoderm cells.

Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells.

The mechanisms underlying the ability of embryonic stem cells (ESCs) to rapidly activate lineage-specific genes during differentiation remain largely unknown. Through multiple CRISPR-activation screens, we discovered human ESCs have pre-established transcriptionally competent chromatin regions (CCRs) that support lineage-specific gene expression at levels comparable to differentiated cells. CCRs reside in the same topological domains as their target genes. They lack typical enhancer-associated histone modifications but show enriched occupancy of pluripotent transcription factors, DNA demethylation factors, and histone deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while HDAC1 family members prevent premature activation. This "push and pull" feature resembles bivalent domains at developmental gene promoters but involves distinct molecular mechanisms. Our study provides new insights into pluripotency regulation and cellular plasticity in development and disease. One sentence summary: We report a class of distal regulatory regions distinct from enhancers that confer human embryonic stem cells with the competence to rapidly activate the expression of lineage-specific genes.

Functional interrogation of twenty type 2 diabetes-associated genes using isogenic hESC-derived ß-like cells.

Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional role of many loci has remained unexplored. In this study, we engineered isogenic knockout human embryonic stem cell (hESC) lines for 20 genes associated with T2D risk. We systematically examined ß-cell differentiation, insulin production and secretion, and survival. We performed RNA-seq and ATAC-seq on hESC-ß cells from each knockout line. Analyses of T2D GWAS signals overlapping with HNF4A-dependent ATAC peaks identified a specific SNP as a likely causal variant. In addition, we performed integrative association analyses and identified four genes ( CP, RNASE1, PCSK1N and GSTA2 ) associated with insulin production, and two genes ( TAGLN3 and DHRS2 ) associated with sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental hESC line, to identify a single likely functional variant at each of 23 T2D GWAS signals.

hPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung's disease.

The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors in vitro and in vivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.

A human iPSC-array-based GWAS identifies a virus susceptibility locus in the NDUFA4 gene and functional variants.

Population-based studies to identify disease-associated risk alleles typically require samples from a large number of individuals. Here, we report a human-induced pluripotent stem cell (hiPSC)-based screening strategy to link human genetics with viral infectivity. A genome-wide association study (GWAS) identified a cluster of single-nucleotide polymorphisms (SNPs) in a cis-regulatory region of the NDUFA4 gene, which was associated with susceptibility to Zika virus (ZIKV) infection. Loss of NDUFA4 led to decreased sensitivity to ZIKV, dengue virus, and SARS-CoV-2 infection. Isogenic hiPSC lines carrying non-risk alleles of SNPs or deletion of the cis-regulatory region lower sensitivity to viral infection. Mechanistic studies indicated that loss/reduction of NDUFA4 causes mitochondrial stress, which leads to the leakage of mtDNA and thereby upregulation of type I interferon signaling. This study provides proof-of-principle for the application of iPSC arrays in GWAS and identifies NDUFA4 as a previously unknown susceptibility locus for viral infection.

Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells.

Precise gene editing in human pluripotent stem cells (hPSCs) holds great promise for studying and potentially treating human diseases. Both prime editing and base editing avoid introducing double strand breaks, but low editing efficiencies make those techniques still an arduous process in hPSCs. Here we report that co-delivering of p53DD, a dominant negative fragment of p53, can greatly enhance prime editing and cytosine base editing efficiencies in generating precise mutations in hPSCs. We further apply PE3 in combination with p53DD to efficiently create multiple isogenic hPSC lines, including lines carrying GBA or LRRK2 mutations associated with Parkinson disease and a LMNA mutation linked to Hutchinson-Gilford progeria syndrome. We also correct GBA and LMNA mutations in the patient-specific iPSCs. Our data show that p53DD improves PE3 efficiency without compromising the genome-wide safety, making it feasible for safe and routine generation of isogenic hPSC lines for disease modeling.

Protocol for the Generation of Human Pluripotent Reporter Cell Lines Using CRISPR/Cas9.

Reporter cell lines based on human pluripotent stem cells (hPSCs) are highly desirable for studying differentiation, lineage tracing, and target cell selection. However, several technical bottlenecks, such as DNA transduction, low homology recombination rate (HDR), and single-cell cloning, have made this effort an arduous process in hPSCs. Here, we provide a step-by-step protocol and practical guide for generating reporter lines in hPSCs via CRISPR/Cas9-mediated HDR. We also elaborate on the process of generating a TBXT-GFP reporter line as an example.

Pre- and peri-implantation Zika virus infection impairs fetal development by targeting trophectoderm cells.

Zika virus (ZIKV) infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the underlying mechanisms remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, at pre- and peri-implantation, because most current ZIKV pregnancy studies have focused on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be infected with ZIKV, and propagate virus causing neural progenitor cell death. These findings are corroborated by the dose-dependent nature of ZIKV susceptibility of hESC-derived trophectoderm cells. Single blastocyst RNA-seq reveals key transcriptional changes upon ZIKV infection, including nervous system development, prior to commitment to the neural lineage. The pregnancy rate of mice is >50% lower in pre-implantation infection than infection at E4.5, demonstrating that pre-implantation ZIKV infection leads to miscarriage. Cumulatively, these data elucidate a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.

Tsc3 regulates SPT amino acid choice in Saccharomyces cerevisiae by promoting alanine in the sphingolipid pathway.

The generation of most sphingolipids (SPLs) starts with condensation between serine and an activated long-chain fatty acid catalyzed by serine palmitoyltransferase (SPT). SPT can also use other amino acids to generate small quantities of noncanonical SPLs. The balance between serine-derived and noncanonical SPLs is pivotal; for example, hereditary sensory and autonomic neuropathy type I results from SPT mutations that cause an abnormal accumulation of alanine-derived SPLs. The regulatory mechanism for SPT amino acid selectivity and physiological functions of noncanonical SPLs are unknown. We investigated SPT selection of amino acid substrates by measuring condensation products of serine and alanine in yeast cultures and SPT use of serine and alanine in a TSC3 knockout model. We identified the Tsc3 subunit of SPT as a regulator of amino acid substrate selectivity by demonstrating its primary function in promoting alanine utilization by SPT and confirmed its requirement for the inhibitory effect of alanine on SPT utilization of serine. Moreover, we observed downstream metabolic consequences to Tsc3 loss: serine influx into the SPL biosynthesis pathway increased through Ypk1-depenedent activation of SPT and ceramide synthases. This Ypk1-dependent activation of serine influx after Tsc3 knockout suggests a potential function for deoxy-sphingoid bases in modulating Ypk1 signaling.

Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae.

Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast Saccharomyces cerevisiae An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin Ki for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH3+ base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.