Digital telomere measurement by long-read sequencing distinguishes healthy aging from disease.

Telomere length is an important biomarker of organismal aging and cellular replicative potential, but existing measurement methods are limited in resolution and accuracy. Here, we deploy digital telomere measurement (DTM) by nanopore sequencing to understand how distributions of human telomere length change with age and disease. We measure telomere attrition and de novo elongation with up to 30 bp resolution in genetically defined populations of human cells, in blood cells from healthy donors and in blood cells from patients with genetic defects in telomere maintenance. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres. In patients with defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease.

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells with different morphological, physiological, and molecular properties that can be classified according to shared properties into cell types. Defining the contribution of each cell type to the computational and cognitive processes that are guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell type taxonomies from mice and humans to define marker genes and enhancers, and to build genetic tools for cortical cell types. Here, we present a large toolkit for selective targeting of cortical populations, including mouse transgenic lines and recombinant adeno-associated virus (AAV) vectors containing genomic enhancers. We report evaluation of fifteen new transgenic driver lines and over 680 different enhancer AAVs covering all major subclasses of cortical cells, with many achieving a high degree of specificity, comparable with existing transgenic lines. We find that the transgenic lines based on marker genes can provide exceptional specificity and completeness of cell type labeling, but frequently require generation of a triple-transgenic cross for best usability/specificity. On the other hand, enhancer AAVs are easy to screen and use, and can be easily modified to express diverse cargo, such as recombinases. However, their use depends on many factors, such as viral titer and route of administration. The tools reported here as well as the scaled process of tool creation provide an unprecedented resource that should enable diverse experimental strategies towards understanding mammalian cortex and brain function.

iSCORE-PD: an isogenic stem cell collection to research Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer the uniique potential to advance our understanding of PD etiology by providing disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this experimental approach, we generated a collection of 55 cell lines genetically engineered to harbor mutations in genes associated with monogenic PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G+FS, SYNJ1 R258Q/FS, VPS13C A444P, VPS13C W395C, GBA1 IVS2+1). All mutations were generated in a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using CRISPR/Cas9 or prime editing-based approaches. We implemented rigorous quality controls, including high density genotyping to detect structural variants and confirm the genomic integrity of each cell line. This systematic approach ensures the high quality of our stem cell collection, highlights differences between conventional CRISPR/Cas9 and prime editing and provides a roadmap for how to generate gene-edited hPSCs collections at scale in an academic setting. We expect that our isogenic stem cell collection will become an accessible platform for the study of PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

Telomerase reverse transcriptase (TERT) promoter mutations (TPMs) are frequently found in different cancer types, including ∼70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma-sequencing datasets, where melanomas with BRAF V600E mutations were more likely to have a CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells.

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Digital telomere measurement by long-read sequencing distinguishes healthy aging from disease.

Telomere length is an important biomarker of organismal aging and cellular replicative potential, but existing measurement methods are limited in resolution and accuracy. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change with age and disease. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human cells, in blood cells from healthy donors and in blood cells from patients with genetic defects in telomere maintenance. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres. In patients with defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease.

Tracing cancer evolution and heterogeneity using Hi-C.

Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples that can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. After validating HiDENSEC with in silico and in vitro controls, we used it to characterize chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and that characterizing these events yields insights into drivers of melanoma progression.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

TERT promoter mutations (TPMs) are frequently found in different cancer types, including approximately 70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres, and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma sequencing data sets, where melanomas with BRAF V600E mutations were more likely to have CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells.

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson's disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge 'search and replace' approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson's disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

Editing TINF2 as a potential therapeutic approach to restore telomere length in dyskeratosis congenita.

Mutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow (BM) failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for BM failure. Here, we endogenously introduced TIN2-DC mutations in human embryonic stem cells (hESCs) and human hematopoietic stem and progenitor cells (HSPCs) to dissect the disease mechanism and identify a gene-editing strategy that rescued the disease phenotypes. The hESCs with the T284R disease mutation exhibited the short telomere phenotype observed in DC patients. Yet, telomeres in mutant hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. Similarly, we introduced TIN2-DC disease variants in human HSPCs to assess the changes in telomere length and proliferative capacity. Lastly, we showed that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSPCs. Our study demonstrates a simple genetic intervention that rescues the TIN2-DC disease phenotype in stem cells and provides a versatile platform to assess the efficacy of potential therapeutic approaches in vivo.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.

Cancer-associated POT1 mutations lead to telomere elongation without induction of a DNA damage response.

Mutations in the shelterin protein POT1 are associated with chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, angiosarcoma, melanoma, and other cancers. These cancer-associated POT1 (caPOT1) mutations are generally heterozygous, missense, or nonsense mutations occurring throughout the POT1 reading frame. Cancers with caPOT1 mutations have elongated telomeres and show increased genomic instability, but which of the two phenotypes promotes tumorigenesis is unclear. We tested the effects of CAS9-engineered caPOT1 mutations in human embryonic and hematopoietic stem cells (hESCs and HSCs, respectively). HSCs with caPOT1 mutations did not show overt telomere damage. In vitro and in vivo competition experiments showed the caPOT1 mutations did not confer a selective disadvantage. Since DNA damage signaling is known to affect the fitness of HSCs, the data argue that caPOT1 mutations do not cause significant telomere damage. Furthermore, hESC lines with caPOT1 mutations showed no detectable telomere damage response while showing consistent telomere elongation. Thus, caPOT1 mutations are likely selected for during cancer progression because of their ability to elongate telomeres and extend the proliferative capacity of the incipient cancer cells.

TINF2 is a haploinsufficient tumor suppressor that limits telomere length.

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.

Analysis of muntjac deer genome and chromatin architecture reveals rapid karyotype evolution.

Closely related muntjac deer show striking karyotype differences. Here we describe chromosome-scale genome assemblies for Chinese and Indian muntjacs, Muntiacus reevesi (2n = 46) and Muntiacus muntjak vaginalis (2n = 6/7), and analyze their evolution and architecture. The genomes show extensive collinearity with each other and with other deer and cattle. We identified numerous fusion events unique to and shared by muntjacs relative to the cervid ancestor, confirming many cytogenetic observations with genome sequence. One of these M. muntjak fusions reversed an earlier fission in the cervid lineage. Comparative Hi-C analysis showed that the chromosome fusions on the M. muntjak lineage altered long-range, three-dimensional chromosome organization relative to M. reevesi in interphase nuclei including A/B compartment structure. This reshaping of multi-megabase contacts occurred without notable change in local chromatin compaction, even near fusion sites. A few genes involved in chromosome maintenance show evidence for rapid evolution, possibly associated with the dramatic changes in karyotype.

Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1.

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1's telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S knockout [KO]), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1L104A/L104A mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1L104A/L104A cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1L104A/L104A shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1L104A/L104A as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.

TERT promoter mutations and telomeres during tumorigenesis.

Telomerase regulation and telomere shortening act as a strong tumor suppressor mechanism in human somatic cells. Point mutations in the promoter of telomerase reverse transcriptase (TERT) are the most frequent non-coding mutation in cancer. These TERT promoter mutations (TPMs) create de novo ETS factor binding sites upstream of the start codon of the gene, which can be bound by different ETS factors. TPMs can occur early during tumorigenesis and are thought to be among the first mutations in melanoma, glioblastoma and hepatocellular carcinoma. Despite their association with increased TERT levels, TPMs do not prohibit telomere shortening and TPM-harboring cancers present with short telomeres. Their short telomere length combined with their high prevalence and specificity for cancer makes TPMs an attractive target for future therapeutic exploitation of telomerase inhibition and telomere deprotection-induced cell death.