Motivation

Most diseases – ranging from cancers to neurodegenerative disorders - are only clinically diagnosed when they have advanced enough to disrupt the body’s normal function. Unfortunately, by this point, the damage inflicted is often irreversible, and traditional clinical treatments can be detrimental to both the surrounding healthy tissue and the overall well-being of the patient. Understanding the early steps in disease onset would enable earlier diagnosis and facilitate the development of more effective targeted treatments.

The Clement lab aims to shift the paradigm of disease study, diagnosis, and treatment by unraveling the genetic and epigenetic mechanisms of disease initiation. We capitalize on three cutting-edge transformative computational and experimental technologies to discover, model, and validate the fundamental mechanisms underpinning disease.

Single-cell technology

First, single-cell technology enables the characterization of individual cell states that are otherwise muddled in studies of heterogenous samples. Recently, methods have been developed to assay the single cell genetic mutation status, gene expression, DNA methylation, chromatin accessibility, and other characteristics that allow us to study and compare cells across samples. We develop methods to analyze single-cell data to develop a clear picture of the cellular signals that drive complex diseases.

CRISPR genome editing

Second, CRISPR technology allows for targeted genomic modification that can be used in research settings as well as clinical treatment. Our lab harnesses the power of CRISPR perturbation to discover and validate fundamental genetic and epigenetic mechanisms underlying diseases. Our lab is also involved in assuring the accuracy of CRISPR technology in research settings and the efficacy and safety of CRISPR applications in clinical settings, by developing novel tools to assess the on- and off-target activity of CRISPR proteins.

Machine learning

Third, the integration of machine learning and big data methods is transforming almost every aspect of modern life. Our lab harnesses these tools to aggregate large datasets and identify subtle signals that are hard to identify using standard statistical methods. In particular, we are interested in the integration of data from different diseases and genomic modalities to discover elements of disease initiation that may be common across diseases.

By synergistically combining these three innovative technologies, we strive to unravel the complex tapestry of disease initiation to pave the way for improved understanding, detection, and treatment of disease. Our work has previously focused on disease initiation in chronic lymphocytic leukemia, but we are eager to collaborate in the study of additional diseases.

Join us on our journey to use cutting-edge genomics to make a lasting impact on human health.

Publications

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2025

Precision enhancement of CAR-NK cells through non-viral engineering and highly multiplexed base editing.
Wang M, Krueger JB, Gilkey AK, Stelljes EM, Kluesner MG, Pomeroy EJ, Skeate JG, Slipek NJ, Lahr WS, Claudio Vazquez PN, Zhao Y, Bell JB, Clement K, Eaton EJ, Laoharawee K, Chang JW, Webber BR, Moriarity BS. J Immunother Cancer. 2025 May 7;13(5). pii: jitc-2024-009560. doi: 10.1136/jitc-2024-009560. PMID 40341025

Plain-language summary We developed a new way to engineer natural killer (NK) cells to fight cancer more effectively using advanced base editing and non-viral gene delivery.

Tags: Non-viral Engineering Base Editing Gene Therapy Cancer Immunotherapy CAR-NK Cells CRISPR

BACKGROUND: Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials. METHODS: Advanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. RESULTS: We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function in vitro and in vivo. Next, we combined multiplex BE with non-viral TcBuster transposon- based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo. CONCLUSIONS: The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell- based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

Genetic and training adaptations in the Haenyeo divers of Jeju, Korea.
Aguilar-Gomez D, Bejder J, Graae J, Ko Y, Vaughn A, Clement K, Tristani-Firouzi M, Lee JY, Nordsborg NB, Nielsen R, Ilardo M. Cell Rep. 2025 Apr 16:115577. doi: 10.1016/j.celrep.2025.115577. PMID 40318638

Plain-language summary We studied the unique genetic and physiological adaptations of the 'Haenyeo' female divers in Korea who hold their breath for long periods in cold water.

Tags: Genomics Physiology Cold Adaptation Hypoxia Human Evolution

Natural selection and relative isolation have shaped the genetics and physiology of unique human populations from Greenland to Tibet. Another such population is the Haenyeo, the all-female Korean divers renowned for their remarkable diving abilities in frigid waters. Apnea diving induces considerable physiological strain, particularly in females diving throughout pregnancy. In this study, we explore the hypothesis that breath-hold diving has shaped physiological and genetic traits in the Haenyeo. We identified pronounced bradycardia during diving, a likely training effect. We paired natural selection and genetic association analyses to investigate adaptive genetic variation that may mitigate the effects of diving on pregnancy through an associated reduction of diastolic blood pressure. Finally, we identified positively selected variation in a gene previously associated with cold water tolerance, which may contribute to reduced hypothermia susceptibility. These findings highlight the importance of traditional diving populations for understanding genetic and physiological adaptation.

Modulating Collagen I Expression in Fibroblasts by CRISPR-Cas9 Base Editing of the Collagen 1A1 Promoter.
Daliri K, Hescheler J, Newby GA, Clement K, Liu DR, Pfannkuche K. Int J Mol Sci. 2025 Mar 26;26(7). pii: ijms26073041. doi: 10.3390/ijms26073041. PMID 40243657

Plain-language summary We used CRISPR base editing to reduce collagen production in fibroblasts, which could be a new way to treat fibrosis and cancer.

Tags: Gene Therapy Fibroblasts Collagen I CRISPR Base Editing CRISPR Fibrosis

Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to- CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.
Zeng J, Nguyen MA, Liu P, da Silva LF, Levesque S, Lin LY, Justus DG, Petri K, Clement K, Porter SN, Verma A, Neri NR, Rosanwo T, Ciuculescu MF, Abriss D, Mintzer E, Maitland SA, Demirci S, Cha HJ, Orkin SH, Tisdale JF, Williams DA, Zhu LJ, Pruett-Miller SM, Pinello L, Joung JK, Pattanayak V, Manis JP, Armant M, Pellin D, Brendel C, Wolfe SA, Bauer DE. Cell Stem Cell. 2025 Feb 6;32(2):191-208.e11. doi: 10.1016/j.stem.2024.11.001. Epub 2024 Dec 12. PMID 39672163

Plain-language summary Scientists have discovered that editing blood stem cells while they are in a resting state, rather than growing them in a laboratory, prevents harmful genetic damage while effectively treating blood disorders like sickle cell disease.

Tags: Fetal hemoglobin (HbF) induction BCL11A erythroid enhancer Genotoxicity CRISPR-Cas9 Hematopoietic Stem and Progenitor Cells (HSPCs) CRISPR

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for beta-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere- distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.

2024

Selective Enhancer Gain-of-Function Deregulates MYC Expression in Multiple Myeloma.
Rahmat M, Clement K, Alberge JB, Sklavenitis-Pistofidis R, Kodgule R, Fulco CP, Heilpern-Mallory D, Nilsson K, Dorfman D, Engreitz JM, Getz G, Pinello L, Ryan RJH, Ghobrial IM. Cancer Res. 2024 Dec 16;84(24):4173-4183. doi: 10.1158/0008-5472.CAN-24-1440. PMID 39312195

Plain-language summary Researchers discovered that multiple myeloma can be driven by an overactive genetic "on-switch" that boosts a cancer-promoting gene called MYC, explaining how the disease progresses even in patients who lack typical large-scale genetic mutations.

Tags: CRISPR interference Gene Therapy Epigenetic regulation Enhancer gain-of-function MYC expression Epigenetics CRISPR Multiple Myeloma

MYC deregulation occurs in the majority of multiple myeloma cases and is associated with progression and worse prognosis. Enhanced MYC expression occurs in about 70% of patients with multiple myeloma, but it is known to be driven by translocation or amplification events in only approximately 40% of myelomas. Here, we used CRISPR interference to uncover an epigenetic mechanism of MYC regulation whereby increased accessibility of a plasma cell-type-specific enhancer leads to increased MYC expression. This native enhancer activity was not associated with enhancer hijacking events but led to specific binding of cMAF, IRF4, and SPIB transcription factors that activated MYC expression in the absence of known genetic aberrations. In addition, focal amplification was another mechanism of activation of this enhancer in approximately 3.4% of patients with multiple myeloma. Together, these findings define an epigenetic mechanism of MYC deregulation in multiple myeloma beyond known translocations or amplifications and point to the importance of noncoding regulatory elements and their associated transcription factor networks as drivers of multiple myeloma progression. Significance: The discovery of a native developmental enhancer that sustains the expression of MYC in a subset of myelomas could help identify predictive biomarkers and therapeutic targets to improve the outcomes of patients with multiple myeloma.

Scalable assessment of genome editing off-targets associated with genetic variants.
Lin J, Nguyen MA, Lin LY, Zeng J, Verma A, Neri NR, da Silva LF, Mucci A, Wolfe S, Shaw KL, Clement K, Brendel C, Pinello L, Pellin D, Bauer DE. bioRxiv. 2024 Jul 25. doi: 10.1101/2024.07.24.605019. PMID 39211178

Plain-language summary Researchers developed a new tool called ABSOLVE-seq to efficiently test whether individual genetic differences might cause unintended DNA changes during gene editing treatments.

Tags: Genetic variants Off-target editing Lentiviral ensemble ABSOLVE-seq Unique Molecular Identifiers (UMIs)

Genome editing with RNA-guided DNA binding factors carries risk of off- target editing at homologous sequences. Genetic variants may introduce sequence changes that increase homology to a genome editing target, thereby increasing risk of off-target editing. Conventional methods to verify candidate off-targets rely on access to cells with genomic DNA carrying these sequences. However, for candidate off-targets associated with genetic variants, appropriate cells for experimental verification may not be available. Here we develop a method, Assessment By Stand-in Off-target LentiViral Ensemble with sequencing (ABSOLVE-seq), to integrate a set of candidate off-target sequences along with unique molecular identifiers (UMIs) in genomes of primary cells followed by clinically relevant gene editor delivery. Gene editing of dozens of candidate off-target sequences may be evaluated in a single experiment with high sensitivity, precision, and power. We provide an open-source pipeline to analyze sequencing data. This approach enables experimental assessment of the influence of human genetic diversity on specificity evaluation during gene editing therapy development.

2023

Generation of mouse models carrying B cell restricted single or multiplexed loss-of-function mutations through CRISPR-Cas9 gene editing.
Ten Hacken E, Gruber M, Hernandez-Sanchez M, Hoffmann GB, Baranowski K, Redd RA, Clement K, Livak K, Wu CJ. STAR Protoc. 2023 Dec 15;4(4):102165. doi: 10.1016/j.xpro.2023.102165. Epub 2023 Sep 18. PMID 37729058

Plain-language summary This protocol describes a method to create specialized mouse models for studying blood cancers by using CRISPR gene editing to selectively disable specific genes only within B cells.

Tags: Multiplexed gene editing Lentiviral transduction CRISPR-Cas9 CRISPR Hematopoietic stem and progenitor cells Lymphoproliferative disorders

Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitro transduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders. For complete details on the use and execution of this protocol, please refer to ten Hacken et al.,(1) ten Hacken et al.,(2) and ten Hacken et al.(3).

Loss-of-function lesions impact B-cell development and fitness but are insufficient to drive CLL in mouse models.
ten Hacken E, Yin S, Redd R, Hernandez Sanchez M, Clement K, Hoffmann GB, Regis FF, Witten E, Li S, Neuberg D, Pinello L, Livak KJ, Wu CJ. Blood Adv. 2023 Aug 22;7(16):4514-4517. doi: 10.1182/bloodadvances.2022009135. PMID 36477552

Plain-language summary We found that while specific gene mutations affect B-cell growth, they are not enough on their own to cause chronic lymphocytic leukemia in mice.

Tags: CLL CLL Modeling In Vivo Editing CRISPR-Cas9 B-cell Development Tumor Suppressors

Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.
Zeng J, Nguyen MA, Liu P, Ferreira da Silva L, Lin LY, Justus DG, Petri K, Clement K, Porter SN, Verma A, Neri NR, Rosanwo T, Ciuculescu MF, Abriss D, Mintzer E, Maitland SA, Demirci S, Tisdale JF, Williams DA, Zhu LJ, Pruett-Miller SM, Pinello L, Joung JK, Pattanayak V, Manis JP, Armant M, Pellin D, Brendel C, Wolfe SA, Bauer DE. bioRxiv. 2023 May 27. doi: 10.1101/2023.05.27.542323. PMID 37292647

Plain-language summary We developed a method to edit blood stem cells without growing them in the lab, reducing genetic damage and improving safety for treating blood disorders.

Tags: Gene Therapy Genotoxicity Sickle Cell Disease In Vivo Gene Editing CRISPR BCL11A HSPCs

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for beta-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.
Ten Hacken E, Sewastianik T, Yin S, Hoffmann GB, Gruber M, Clement K, Penter L, Redd RA, Ruthen N, Hergalant S, Sholokhova A, Fell G, Parry EM, Broseus J, Guieze R, Lucas F, Hernandez-Sanchez M, Baranowski K, Southard J, Joyal H, Billington L, Regis FFD, Witten E, Uduman M, Knisbacher BA, Li S, Lyu H, Vaisitti T, Deaglio S, Inghirami G, Feugier P, Stilgenbauer S, Tausch E, Davids MS, Getz G, Livak KJ, Bozic I, Neuberg DS, Carrasco RD, Wu CJ. Blood Cancer Discov. 2023 Mar 1;4(2):150-169. doi: 10.1158/2643-3230.BCD-22-0082. PMID 36468984

Plain-language summary We created a mouse model to study how chronic lymphocytic leukemia transforms into a more aggressive lymphoma, identifying key genetic drivers.

Tags: Tumor Evolution Gene Therapy CLL Mouse Models In Vivo CRISPR CRISPR Richter Transformation

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

JAK-STAT Signaling in Inflammatory Breast Cancer Enables Chemotherapy- Resistant Cell States.
Stevens LE, Peluffo G, Qiu X, Temko D, Fassl A, Li Z, Trinh A, Seehawer M, Jovanovic B, Aleckovic M, Wilde CM, Geck RC, Shu S, Kingston NL, Harper NW, Almendro V, Pyke AL, Egri SB, Papanastasiou M, Clement K, Zhou N, Walker S, Salas J, Park SY, Frank DA, Meissner A, Jaffe JD, Sicinski P, Toker A, Michor F, Long HW, Overmoyer BA, Polyak K. Cancer Res. 2023 Jan 18;83(2):264-284. doi: 10.1158/0008-5472.CAN-22-0423. PMID 36409824

Plain-language summary We identified a signaling pathway that helps inflammatory breast cancer cells resist chemotherapy and showed that blocking it improves treatment.

Tags: Chemoresistance JAK-STAT Signaling Gene Therapy Inflammatory Breast Cancer Single-Cell Analysis Epigenetics Targeted Therapy

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC. SIGNIFICANCE: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors.

2022

CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells.
Petri K, Zhang W, Ma J, Schmidts A, Lee H, Horng JE, Kim DY, Kurt IC, Clement K, Hsu JY, Pinello L, Maus MV, Joung JK, Yeh JJ. Nat Biotechnol. 2022 Feb;40(2):189-193. doi: 10.1038/s41587-021-00901-y. Epub 2021 Apr 29. PMID 33927418

Plain-language summary We demonstrated that 'prime editing' works in zebrafish and human cells using purified protein complexes, offering a new way to make precise DNA changes.

Tags: Ribonucleoproteins (RNP) Zebrafish Precision Medicine Germline Editing CRISPR Prime Editing

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.

2021

A Code of Ethics for Gene Drive Research.
Annas GJ, Beisel CL, Clement K, Crisanti A, Francis S, Galardini M, Galizi R, Grunewald J, Immobile G, Khalil AS, Muller R, Pattanayak V, Petri K, Paul L, Pinello L, Simoni A, Taxiarchi C, Joung JK. CRISPR J. 2021 Feb;4(1):19-24. doi: 10.1089/crispr.2020.0096. Epub 2021 Feb 10. PMID 33571044

Plain-language summary We proposed a code of ethics to guide research on gene drives, ensuring this powerful technology is developed responsibly.

Tags: Public Health Gene Drive Bioethics Ecological Risk Research Guidelines

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.

Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy.
Kretzmer H, Biran A, Purroy N, Lemvigh CK, Clement K, Gruber M, Gu H, Rassenti L, Mohammad AW, Lesnick C, Slager SL, Braggio E, Shanafelt TD, Kay NE, Fernandes SM, Brown JR, Wang L, Li S, Livak KJ, Neuberg DS, Klages S, Timmermann B, Kipps TJ, Campo E, Gnirke A, Wu CJ, Meissner A. Blood Cancer Discov. 2021 Jan;2(1):54-69. doi: 10.1158/2643-3230.BCD-19-0058. Epub 2020 Dec 3. PMID 33604581

Plain-language summary We mapped the DNA methylation changes in early-stage chronic lymphocytic leukemia and found they are established before the disease becomes aggressive.

Tags: CLL Epigenetics MBL Epigenetic Evolution DNA Methylation Pre-neoplastic States

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in pre-neoplastic monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significantly differentially methylated regions in the high- count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and post-therapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state, and a distinct expression profile together with MBL cells compared to normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically-driven growth dynamics and together with its persistent presence suggests a central role in the normal-to-cancer transition.

2020

High throughput single-cell detection of multiplex CRISPR-edited gene modifications.
Ten Hacken E, Clement K, Li S, Hernandez-Sanchez M, Redd R, Wang S, Ruff D, Gruber M, Baranowski K, Jacob J, Flynn J, Jones KW, Neuberg D, Livak KJ, Pinello L, Wu CJ. Genome Biol. 2020 Oct 20;21(1):266. doi: 10.1186/s13059-020-02174-1. PMID 33081820

Plain-language summary We used a high-throughput single-cell method to analyze how CRISPR edits genes, revealing complex mutational patterns.

Tags: Genomics CLL Mutational Profiling CRISPR-Cas9 Single-Cell Single-Cell Analysis CRISPR Multiplexed Editing

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.

Distinct evolutionary paths in chronic lymphocytic leukemia during resistance to the graft-versus-leukemia effect.
Bachireddy P, Ennis C, Nguyen VN, Gohil SH, Clement K, Shukla SA, Forman J, Barkas N, Freeman S, Bavli N, Elagina L, Leshchiner I, Mohammad AW, Mathewson ND, Keskin DB, Rassenti LZ, Kipps TJ, Brown JR, Getz G, Ho VT, Gnirke A, Neuberg D, Soiffer RJ, Ritz J, Alyea EP, Kharchenko PV, Wu CJ. Sci Transl Med. 2020 Sep 16;12(561). pii: 12/561/eabb7661. doi: 10.1126/scitranslmed.abb7661. PMID 32938797

Plain-language summary We discovered that leukemia relapse after stem cell transplant follows distinct evolutionary paths depending on when it occurs.

Tags: CLL Single-Cell CLL Relapse Epigenetics Epigenetic Evolution Allo-HSCT Single-Cell Transcriptomics Graft-versus-Leukemia

Leukemic relapse remains a major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive hematologic malignancies. The basis for relapse of advanced lymphoid malignancies remains incompletely understood and may involve escape from the graft-versus-leukemia (GvL) effect. We hypothesized that for patients with chronic lymphocytic leukemia (CLL) treated with allo-HSCT, leukemic cell-intrinsic features influence transplant outcomes by directing the evolutionary trajectories of CLL cells. Integrated genetic, transcriptomic, and epigenetic analyses of CLL cells from 10 patients revealed that the clinical kinetics of post-HSCT relapse are shaped by distinct molecular dynamics. Early relapses after allo-HSCT exhibited notable genetic stability; single CLL cell transcriptional analysis demonstrated a cellular heterogeneity that was static over time. In contrast, CLL cells relapsing late after allo-HSCT displayed notable genetic evolution and evidence of neoantigen depletion, consistent with marked single-cell transcriptional shifts that were unique to each patient. We observed a greater rate of epigenetic change for late relapses not seen in early relapses or relapses after chemotherapy alone, suggesting that the selection pressures of the GvL bottleneck are unlike those imposed by chemotherapy. No selective advantage for human leukocyte antigen (HLA) loss was observed, even when present in pretransplant subpopulations. Gain of stem cell modules was a common signature associated with leukemia relapse regardless of posttransplant relapse kinetics. These data elucidate the biological pathways that underlie GvL resistance and posttransplant relapse.

Technologies and Computational Analysis Strategies for CRISPR Applications.
Clement K, Hsu JY, Canver MC, Joung JK, Pinello L. Mol Cell. 2020 Jul 2;79(1):11-29. doi: 10.1016/j.molcel.2020.06.012. PMID 32619467

Plain-language summary This review covers current CRISPR technologies and the computational tools used to analyze the data they generate.

Tags: Data Analysis Gene Therapy Off-target Analysis Computational Biology CRISPR Screens Epigenetics CRISPR Gene Editing

The CRISPR-Cas system offers a programmable platform for eukaryotic genome and epigenome editing. The ability to perform targeted genetic and epigenetic perturbations enables researchers to perform a variety of tasks, ranging from investigating questions in basic biology to potentially developing novel therapeutics for the treatment of disease. While CRISPR systems have been engineered to target DNA and RNA with increased precision, efficiency, and flexibility, assays to identify off- target editing are becoming more comprehensive and sensitive. Furthermore, techniques to perform high-throughput genome and epigenome editing can be paired with a variety of readouts and are uncovering important cellular functions and mechanisms. These technological advances drive and are driven by accompanying computational approaches. Here, we briefly present available CRISPR technologies and review key computational advances and considerations for various CRISPR applications. In particular, we focus on the analysis of on- and off- target editing and CRISPR pooled screen data.

Therapeutic base editing of human hematopoietic stem cells.
Zeng J, Wu Y, Ren C, Bonanno J, Shen AH, Shea D, Gehrke JM, Clement K, Luk K, Yao Q, Kim R, Wolfe SA, Manis JP, Pinello L, Joung JK, Bauer DE. Nat Med. 2020 Apr;26(4):535-541. doi: 10.1038/s41591-020-0790-y. Epub 2020 Mar 16. PMID 32284612

Plain-language summary We showed that 'base editing' can correct genetic mutations in human blood stem cells to treat sickle cell disease and beta-thalassemia.

Tags: Base Editing Gene Therapy Sickle Cell Disease Beta-Thalassemia CRISPR HSPCs

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and beta-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self- renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC- targeted therapeutic genome modification.

2019

Assessment of computational methods for the analysis of single-cell ATAC- seq data.
Chen H, Lareau C, Andreani T, Vinyard ME, Garcia SP, Clement K, Andrade-Navarro MA, Buenrostro JD, Pinello L. Genome Biol. 2019 Nov 18;20(1):241. doi: 10.1186/s13059-019-1854-5. PMID 31739806

Plain-language summary We compared different software methods for analyzing single-cell chromatin accessibility data to identify the best tools.

Tags: scATAC-seq Single-Cell Epigenetics Benchmarking Single-Cell Epigenomics Computational Methods Chromatin Accessibility

BACKGROUND: Recent innovations in single-cell Assay for Transposase Accessible Chromatin using sequencing (scATAC-seq) enable profiling of the epigenetic landscape of thousands of individual cells. scATAC-seq data analysis presents unique methodological challenges. scATAC-seq experiments sample DNA, which, due to low copy numbers (diploid in humans), lead to inherent data sparsity (1-10% of peaks detected per cell) compared to transcriptomic (scRNA-seq) data (10-45% of expressed genes detected per cell). Such challenges in data generation emphasize the need for informative features to assess cell heterogeneity at the chromatin level. RESULTS: We present a benchmarking framework that is applied to 10 computational methods for scATAC-seq on 13 synthetic and real datasets from different assays, profiling cell types from diverse tissues and organisms. Methods for processing and featurizing scATAC-seq data were compared by their ability to discriminate cell types when combined with common unsupervised clustering approaches. We rank evaluated methods and discuss computational challenges associated with scATAC-seq analysis including inherently sparse data, determination of features, peak calling, the effects of sequencing coverage and noise, and clustering performance. Running times and memory requirements are also discussed. CONCLUSIONS: This reference summary of scATAC-seq methods offers recommendations for best practices with consideration for both the non-expert user and the methods developer. Despite variation across methods and datasets, SnapATAC, Cusanovich2018, and cisTopic outperform other methods in separating cell populations of different coverages and noise levels in both synthetic and real datasets. Notably, SnapATAC is the only method able to analyze a large dataset (> 80,000 cells).

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis.
Di Stefano B, Luo EC, Haggerty C, Aigner S, Charlton J, Brumbaugh J, Ji F, Rabano Jimenez I, Clowers KJ, Huebner AJ, Clement K, Lipchina I, de Kort MAC, Anselmo A, Pulice J, Gerli MFM, Gu H, Gygi SP, Sadreyev RI, Meissner A, Yeo GW, Hochedlinger K. Cell Stem Cell. 2019 Nov 7;25(5):622-638.e13. doi: 10.1016/j.stem.2019.08.018. Epub 2019 Oct 3. PMID 31588046

Plain-language summary We found that the protein DDX6 controls the balance between stem cell self-renewal and differentiation by regulating mRNA stability.

Tags: Stem Cell Pluripotency P-bodies Epigenetics Reprogramming DDX6 Post-transcriptional Regulation

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate- instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.

Epigenetic evolution and lineage histories of chronic lymphocytic leukaemia.
Gaiti F, Chaligne R, Gu H, Brand RM, Kothen-Hill S, Schulman RC, Grigorev K, Risso D, Kim KT, Pastore A, Huang KY, Alonso A, Sheridan C, Omans ND, Biederstedt E, Clement K, Wang L, Felsenfeld JA, Bhavsar EB, Aryee MJ, Allan JN, Furman R, Gnirke A, Wu CJ, Meissner A, Landau DA. Nature. 2019 May;569(7757):576-580. doi: 10.1038/s41586-019-1198-z. Epub 2019 May 15. PMID 31092926

Plain-language summary We used epigenetic marks to trace the history and evolution of cancer cells in patients with chronic lymphocytic leukemia.

Tags: Phylogenetics CLL Lineage Tracing Single-Cell Epigenetics Epigenetic Evolution Single-Cell Methylomics

Genetic and epigenetic intra-tumoral heterogeneity cooperate to shape the evolutionary course of cancer(1). Chronic lymphocytic leukaemia (CLL) is a highly informative model for cancer evolution as it undergoes substantial genetic diversification and evolution after therapy(2,3). The CLL epigenome is also an important disease-defining feature(4,5), and growing populations of cells in CLL diversify by stochastic changes in DNA methylation known as epimutations(6). However, previous studies using bulk sequencing methods to analyse the patterns of DNA methylation were unable to determine whether epimutations affect CLL populations homogeneously. Here, to measure the epimutation rate at single-cell resolution, we applied multiplexed single-cell reduced-representation bisulfite sequencing to B cells from healthy donors and patients with CLL. We observed that the common clonal origin of CLL results in a consistently increased epimutation rate, with low variability in the cell-to-cell epimutation rate. By contrast, variable epimutation rates across healthy B cells reflect diverse evolutionary ages across the trajectory of B cell differentiation, consistent with epimutations serving as a molecular clock. Heritable epimutation information allowed us to reconstruct lineages at high-resolution with single-cell data, and to apply this directly to patient samples. The CLL lineage tree shape revealed earlier branching and longer branch lengths than in normal B cells, reflecting rapid drift after the initial malignant transformation and a greater proliferative history. Integration of single-cell bisulfite sequencing analysis with single-cell transcriptomes and genotyping confirmed that genetic subclones mapped to distinct clades, as inferred solely on the basis of epimutation information. Finally, to examine potential lineage biases during therapy, we profiled serial samples during ibrutinib-associated lymphocytosis, and identified clades of cells that were preferentially expelled from the lymph node after treatment, marked by distinct transcriptional profiles. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of CLL and its evolution with therapy.

Highly efficient therapeutic gene editing of human hematopoietic stem cells.
Wu Y, Zeng J, Roscoe BP, Liu P, Yao Q, Lazzarotto CR, Clement K, Cole MA, Luk K, Baricordi C, Shen AH, Ren C, Esrick EB, Manis JP, Dorfman DM, Williams DA, Biffi A, Brugnara C, Biasco L, Brendel C, Pinello L, Tsai SQ, Wolfe SA, Bauer DE. Nat Med. 2019 May;25(5):776-783. doi: 10.1038/s41591-019-0401-y. Epub 2019 Mar 25. PMID 30911135

Plain-language summary We achieved highly efficient gene editing in human blood stem cells to treat hemoglobin disorders like sickle cell disease.

Tags: Gene Therapy Sickle Cell Disease CRISPR-Cas9 Beta-Thalassemia CRISPR BCL11A HSPCs

Re-expression of the paralogous gamma-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe beta-globin disorders sickle cell disease (SCD) and beta-thalassemia by induction of fetal hemoglobin (HbF, alpha2gamma2)(1). Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells(2-6). CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal gamma-globin. We optimize conditions for selection-free on-target editing in patient- derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology- mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with beta-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

CRISPResso2 provides accurate and rapid genome editing sequence analysis.
Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, Cole MA, Liu DR, Joung JK, Bauer DE, Pinello L. Nat Biotechnol. 2019 Mar;37(3):224-226. doi: 10.1038/s41587-019-0032-3. PMID 30809026

Plain-language summary We benchmarked computational methods for analyzing single-cell ATAC-seq data to guide researchers in choosing the right tools.

Tags: scATAC-seq Epigenomics Computational Biology Single-Cell Single-Cell Analysis CRISPR Benchmarking

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.
Kleinstiver BP, Sousa AA, Walton RT, Tak YE, Hsu JY, Clement K, Welch MM, Horng JE, Malagon-Lopez J, Scarfo I, Maus MV, Pinello L, Aryee MJ, Joung JK. Nat Biotechnol. 2019 Mar;37(3):276-282. doi: 10.1038/s41587-018-0011-0. Epub 2019 Feb 11. PMID 30742127

Plain-language summary We released CRISPResso2, an improved software tool for analyzing and quantifying genome editing outcomes from sequencing data.

Tags: Genome Editing Analysis Sequencing Data Software Quantification Epigenetics CRISPResso2 CRISPR

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases(1) has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)(2). To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants(3) to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.

2018

CRISPR-SURF: discovering regulatory elements by deconvolution of CRISPR tiling screen data.
Hsu JY, Fulco CP, Cole MA, Canver MC, Pellin D, Sher F, Farouni R, Clement K, Guo JA, Biasco L, Orkin SH, Engreitz JM, Lander ES, Joung JK, Bauer DE, Pinello L. Nat Methods. 2018 Dec;15(12):992-993. doi: 10.1038/s41592-018-0225-6. PMID 30504875

Plain-language summary We engineered new variants of the Cas12a enzyme that can target more of the genome with higher precision.

Tags: Base Editing Expanded PAM Cas12a/Cpf1 High-Fidelity Protein Engineering CRISPR

Targets and genomic constraints of ectopic Dnmt3b expression.
Zhang Y, Charlton J, Karnik R, Beerman I, Smith ZD, Gu H, Boyle P, Mi X, Clement K, Pop R, Gnirke A, Rossi DJ, Meissner A. Elife. 2018 Nov 23;7. pii: 40757. doi: 10.7554/eLife.40757. PMID 30468428

Plain-language summary We developed CRISPR-SURF, a computational method to discover regulatory elements in the non-coding genome using CRISPR screens.

Tags: CRISPR Screens Epigenetics Regulatory Elements CRISPR Non-coding Genome Deconvolution CRISPR-SURF

DNA methylation plays an essential role in mammalian genomes and expression of the responsible enzymes is tightly controlled. Deregulation of the de novo DNA methyltransferase DNMT3B is frequently observed across cancer types, yet little is known about its ectopic genomic targets. Here, we used an inducible transgenic mouse model to delineate rules for abnormal DNMT3B targeting, as well as the constraints of its activity across different cell types. Our results explain the preferential susceptibility of certain CpG islands to aberrant methylation and point to transcriptional state and the associated chromatin landscape as the strongest predictors. Although DNA methylation and H3K27me3 are usually non-overlapping at CpG islands, H3K27me3 can transiently co-occur with DNMT3B-induced DNA methylation. Our genome-wide data combined with ultra- deep locus-specific bisulfite sequencing suggest a distributive activity of ectopically expressed Dnmt3b that leads to discordant CpG island hypermethylation and provides new insights for interpreting the cancer methylome.

Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons.
Ichida JK, Staats KA, Davis-Dusenbery BN, Clement K, Galloway KE, Babos KN, Shi Y, Son EY, Kiskinis E, Atwater N, Gu H, Gnirke A, Meissner A, Eggan K. Development. 2018 Nov 21;145(22). pii: dev.168617. doi: 10.1242/dev.168617. PMID 30337375

Plain-language summary We investigated how the enzyme DNMT3B targets DNA for methylation and how this process goes wrong in cancer.

Tags: Cancer Epigenetics CpG Islands DNMT3B Epigenetics Ectopic Expression DNA Methylation

Advances in stem cell science allow the production of different cell types in vitro either through the recapitulation of developmental processes, often termed 'directed differentiation', or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between in vitro-derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code and synaptic gene expression between lineage- converted and directed differentiation motor neurons that affect their utility in translational studies.

An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities.
Gehrke JM, Cervantes O, Clement MK, Wu Y, Zeng J, Bauer DE, Pinello L, Joung JK. Nat Biotechnol. 2018 Nov;36(10):977-982. doi: 10.1038/nbt.4199. Epub 2018 Jul 30. PMID 30059493

Plain-language summary We compared motor neurons made from stem cells to real ones and found they are molecularly similar but have some specific differences.

Tags: Motor Neurons Direct Reprogramming iPSCs Methylomics CRISPR Transcriptomics

Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.

In vivo CRISPR editing with no detectable genome-wide off-target mutations.
Akcakaya P, Bobbin ML, Guo JA, Malagon-Lopez J, Clement K, Garcia SP, Fellows MD, Porritt MJ, Firth MA, Carreras A, Baccega T, Seeliger F, Bjursell M, Tsai SQ, Nguyen NT, Nitsch R, Mayr LM, Pinello L, Bohlooly-Y M, Aryee MJ, Maresca M, Joung JK. Nature. 2018 Sep;561(7723):416-419. doi: 10.1038/s41586-018-0500-9. Epub 2018 Sep 12. PMID 30209390

Plain-language summary We engineered a more precise 'base editor' that reduces unwanted mutations in nearby DNA.

Tags: Base Editing Gene Therapy APOBEC3A Precision Editing Off-target Effects CRISPR Protein Engineering

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications(1-6) but identifying unwanted off-target mutations is important for clinical translation(7). A well- validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.
Di Stefano B, Ueda M, Sabri S, Brumbaugh J, Huebner AJ, Sahakyan A, Clement K, Clowers KJ, Erickson AR, Shioda K, Gygi SP, Gu H, Shioda T, Meissner A, Takashima Y, Plath K, Hochedlinger K. Nat Methods. 2018 Sep;15(9):732-740. doi: 10.1038/s41592-018-0104-1. Epub 2018 Aug 20. PMID 30127506

Plain-language summary We developed VIVO, a method to reliably detect off-target CRISPR mutations in living organisms.

Tags: VIVO Gene Therapy In Vivo Editing Mouse Models Epigenetics CRISPR Safety Off-target Detection CRISPR

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Prospective Isolation of Poised iPSC Intermediates Reveals Principles of Cellular Reprogramming.
Schwarz BA, Cetinbas M, Clement K, Walsh RM, Cheloufi S, Gu H, Langkabel J, Kamiya A, Schorle H, Meissner A, Sadreyev RI, Hochedlinger K. Cell Stem Cell. 2018 Aug 2;23(2):289-305.e5. doi: 10.1016/j.stem.2018.06.013. Epub 2018 Jul 12. PMID 30017590

Plain-language summary We found a way to grow human embryonic stem cells that makes them more stable and less prone to genetic defects.

Tags: MEK Inhibition Naive hESCs Epigenetics Genomic Stability Culture Conditions

Cellular reprogramming converts differentiated cells into induced pluripotent stem cells (iPSCs). However, this process is typically very inefficient, complicating mechanistic studies. We identified and molecularly characterized rare, early intermediates poised to reprogram with up to 95% efficiency, without perturbing additional genes or pathways, during iPSC generation from mouse embryonic fibroblasts. Analysis of these cells uncovered transcription factors (e.g., Tfap2c and Bex2) that are important for reprogramming but dispensable for pluripotency maintenance. Additionally, we observed striking patterns of chromatin hyperaccessibility at pluripotency loci, which preceded gene expression in poised intermediates. Finally, inspection of these hyperaccessible regions revealed an early wave of DNA demethylation that is uncoupled from de novo methylation of somatic regions late in reprogramming. Our study underscores the importance of investigating rare intermediates poised to produce iPSCs, provides insights into reprogramming mechanisms, and offers a valuable resource for the dissection of transcriptional and epigenetic dynamics intrinsic to cell fate change.

AmpUMI: design and analysis of unique molecular identifiers for deep amplicon sequencing.
Clement K, Farouni R, Bauer DE, Pinello L. Bioinformatics. 2018 Jul 1;34(13):i202-i210. doi: 10.1093/bioinformatics/bty264. PMID 29949956

Plain-language summary We created AmpUMI, a software tool to design and analyze unique molecular identifiers for more accurate DNA sequencing.

Tags: Software Single-Cell Error Correction UMIs Amplicon Sequencing NGS

Motivation: Unique molecular identifiers (UMIs) are added to DNA fragments before PCR amplification to discriminate between alleles arising from the same genomic locus and sequencing reads produced by PCR amplification. While computational methods have been developed to take into account UMI information in genome-wide and single-cell sequencing studies, they are not designed for modern amplicon-based sequencing experiments, especially in cases of high allelic diversity. Importantly, no guidelines are provided for the design of optimal UMI length for amplicon-based sequencing experiments. Results: Based on the total number of DNA fragments and the distribution of allele frequencies, we present a model for the determination of the minimum UMI length required to prevent UMI collisions and reduce allelic distortion. We also introduce a user- friendly software tool called AmpUMI to assist in the design and the analysis of UMI-based amplicon sequencing studies. AmpUMI provides quality control metrics on frequency and quality of UMIs, and trims and deduplicates amplicon sequences with user specified parameters for use in downstream analysis. Availability and implementation: AmpUMI is open- source and freely available at http://github.com/pinellolab/AmpUMI.

An Intermediate Pluripotent State Controlled by MicroRNAs Is Required for the Naive-to-Primed Stem Cell Transition.
Du P, Pirouz M, Choi J, Huebner AJ, Clement K, Meissner A, Hochedlinger K, Gregory RI. Cell Stem Cell. 2018 Jun 1;22(6):851-864.e5. doi: 10.1016/j.stem.2018.04.021. Epub 2018 May 24. PMID 29804889

Plain-language summary We identified a key protein, ISY1, that uses microRNAs to control the transition of stem cells from a naive to a primed state.

Tags: Pluripotency MicroRNAs Stem Cells Naive-to-Primed Transition ISY1

The embryonic stem cell (ESC) transition from naive to primed pluripotency is marked by major changes in cellular properties and developmental potential. ISY1 regulates microRNA (miRNA) biogenesis, yet its role and relevance to ESC biology remain unknown. Here, we find that highly dynamic ISY1 expression during the naive-to-primed ESC transition defines a specific phase of "poised" pluripotency characterized by distinct miRNA and mRNA transcriptomes and widespread poised cell contribution to mouse chimeras. Loss- and gain-of-function experiments reveal that ISY1 promotes exit from the naive state and is necessary and sufficient to induce and maintain poised pluripotency, and that persistent ISY1 overexpression inhibits the transition from the naive to the primed state. We identify a large subset of ISY1-dependent miRNAs that can rescue the inability of miRNA-deficient ESCs to establish the poised state and transition to the primed state. Thus, dynamic ISY1 regulates poised pluripotency through miRNAs to control ESC fate.

Cancer-Germline Antigen Expression Discriminates Clinical Outcome to CTLA-4 Blockade.
Shukla SA, Bachireddy P, Schilling B, Galonska C, Zhan Q, Bango C, Langer R, Lee PC, Gusenleitner D, Keskin DB, Babadi M, Mohammad A, Gnirke A, Clement K, Cartun ZJ, Van Allen EM, Miao D, Huang Y, Snyder A, Merghoub T, Wolchok JD, Garraway LA, Meissner A, Weber JS, Hacohen N, Neuberg D, Potts PR, Murphy GF, Lian CG, Schadendorf D, Hodi FS, Wu CJ. Cell. 2018 Apr 19;173(3):624-633.e8. doi: 10.1016/j.cell.2018.03.026. Epub 2018 Apr 12. PMID 29656892

Plain-language summary We found a gene signature in melanoma patients that predicts whether they will respond to specific immunotherapy drugs.

Tags: Melanoma Gene Therapy Immunotherapy Resistance Cancer-Germline Antigens CTLA-4 Blockade MAGE-A

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.
Cesana M, Guo MH, Cacchiarelli D, Wahlster L, Barragan J, Doulatov S, Vo LT, Salvatori B, Trapnell C, Clement K, Cahan P, Tsanov KM, Sousa PM, Tazon-Vega B, Bolondi A, Giorgi FM, Califano A, Rinn JL, Meissner A, Hirschhorn JN, Daley GQ. Cell Stem Cell. 2018 Apr 5;22(4):575-588.e7. doi: 10.1016/j.stem.2018.03.012. PMID 29625070

Plain-language summary We discovered how alternative splicing of the HMGA2 gene regulates the development of human blood stem cells.

Tags: CLK3 HSCs Developmental Biology HMGA2 Alternative Splicing

While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs.

Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".
Lareau CA, Clement K, Hsu JY, Pattanayak V, Joung JK, Aryee MJ, Pinello L. Nat Methods. 2018 Apr;15(4):238-239. doi: 10.1038/nmeth.4541. Epub 2018 Mar 30. PMID 29600992

Plain-language summary A response to a previous paper discussing the frequency of unexpected mutations after CRISPR editing.

Tags: Genomics Off-target Mutations Gene Therapy Scientific Debate In Vivo Editing CRISPR Safety CRISPR

Global delay in nascent strand DNA methylation.
Charlton J, Downing TL, Smith ZD, Gu H, Clement K, Pop R, Akopian V, Klages S, Santos DP, Tsankov AM, Timmermann B, Ziller MJ, Kiskinis E, Gnirke A, Meissner A. Nat Struct Mol Biol. 2018 Apr;25(4):327-332. doi: 10.1038/s41594-018-0046-4. Epub 2018 Mar 12. PMID 29531288

Plain-language summary We showed that there is a delay in copying DNA methylation patterns when cells divide, which is important for maintaining cell identity.

Tags: Epigenetic Inheritance Cell Cycle DNA Replication Epigenetics Stem Cells DNA Methylation

Cytosine methylation is widespread among organisms and essential for mammalian development. In line with early postulations of an epigenetic role in gene regulation, symmetric CpG methylation can be mitotically propagated over many generations with extraordinarily high fidelity. Here, we combine BrdU labeling and immunoprecipitation with genome-wide bisulfite sequencing to explore the inheritance of cytosine methylation onto newly replicated DNA in human cells. Globally, we observe a pronounced lag between the copying of genetic and epigenetic information in embryonic stem cells that is reconsolidated within hours to accomplish faithful mitotic transmission. Populations of arrested cells show a global reduction of lag-induced intermediate CpG methylation when compared to proliferating cells, whereas sites of transcription factor engagement appear cell-cycle invariant. Alternatively, the cancer cell line HCT116 preserves global epigenetic heterogeneity independently of cell-cycle arrest. Taken together, our data suggest that heterogeneous methylation largely reflects asynchronous proliferation, but is intrinsic to actively engaged cis-regulatory elements and cancer.

Genome-wide tracking of dCas9-methyltransferase footprints.
Galonska C, Charlton J, Mattei AL, Donaghey J, Clement K, Gu H, Mohammad AW, Stamenova EK, Cacchiarelli D, Klages S, Timmermann B, Cantz T, Scholer HR, Gnirke A, Ziller MJ, Meissner A. Nat Commun. 2018 Feb 9;9(1):597. doi: 10.1038/s41467-017-02708-5. PMID 29426832

Plain-language summary We tracked where a modified CRISPR tool travels in the genome to understand how it can be used for epigenetic editing.

Tags: Epigenetic Editing Genomic Footprinting dCas9 ChIP-seq Epigenetics CRISPR DNA Methylation

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

Genetic determinants and epigenetic effects of pioneer-factor occupancy.
Donaghey J, Thakurela S, Charlton J, Chen JS, Smith ZD, Gu H, Pop R, Clement K, Stamenova EK, Karnik R, Kelley DR, Gifford CA, Cacchiarelli D, Rinn JL, Gnirke A, Ziller MJ, Meissner A. Nat Genet. 2018 Feb;50(2):250-258. doi: 10.1038/s41588-017-0034-3. Epub 2018 Jan 22. PMID 29358654

Plain-language summary We studied 'pioneer' transcription factors to understand how they can access closed chromatin to turn on genes.

Tags: Epigenetics FOXA2 Pioneer Factors GATA4 Transcription Factors Chromatin Accessibility

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.

2017

Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer.
Smith ZD, Shi J, Gu H, Donaghey J, Clement K, Cacchiarelli D, Gnirke A, Michor F, Meissner A. Nature. 2017 Sep 28;549(7673):543-547. doi: 10.1038/nature23891. Epub 2017 Sep 20. PMID 28959968

Plain-language summary We analyzed how the placenta acquires its unique DNA methylation pattern and how this relates to cancer.

Tags: Cancer Methylome Development Epigenetic Restriction Placenta Epigenetics DNA Methylation

In mammals, the canonical somatic DNA methylation landscape is established upon specification of the embryo proper and subsequently disrupted within many cancer types. However, the underlying mechanisms that direct this genome-scale transformation remain elusive, with no clear model for its systematic acquisition or potential developmental utility. Here, we analysed global remethylation from the mouse preimplantation embryo into the early epiblast and extraembryonic ectoderm. We show that these two states acquire highly divergent genomic distributions with substantial disruption of bimodal, CpG density- dependent methylation in the placental progenitor. The extraembryonic epigenome includes specific de novo methylation at hundreds of embryonically protected CpG island promoters, particularly those that are associated with key developmental regulators and are orthologously methylated across most human cancer types. Our data suggest that the evolutionary innovation of extraembryonic tissues may have required co- option of DNA methylation-based suppression as an alternative to regulation by Polycomb-group proteins, which coordinate embryonic germ- layer formation in response to extraembryonic cues. Moreover, we establish that this decision is made deterministically, downstream of promiscuously used-and frequently oncogenic-signalling pathways, via a novel combination of epigenetic cofactors. Methylation of developmental gene promoters during tumorigenesis may therefore reflect the misappropriation of an innate trajectory and the spontaneous reacquisition of a latent, developmentally encoded epigenetic landscape.

Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.
Choi J, Huebner AJ, Clement K, Walsh RM, Savol A, Lin K, Gu H, Di Stefano B, Brumbaugh J, Kim SY, Sharif J, Rose CM, Mohammad A, Odajima J, Charron J, Shioda T, Gnirke A, Gygi S, Koseki H, Sadreyev RI, Xiao A, Meissner A, Hochedlinger K. Nature. 2017 Aug 10;548(7666):219-223. doi: 10.1038/nature23274. Epub 2017 Jul 26. PMID 28746311

Plain-language summary We found that prolonged exposure to certain chemicals used to grow stem cells can impair their ability to develop properly.

Tags: Imprinting Gene Therapy Naive Pluripotency MEK Inhibition Epigenetics Stem Cell Culture Epigenetic Instability

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells.
Choi J, Clement K, Huebner AJ, Webster J, Rose CM, Brumbaugh J, Walsh RM, Lee S, Savol A, Etchegaray JP, Gu H, Boyle P, Elling U, Mostoslavsky R, Sadreyev R, Park PJ, Gygi SP, Meissner A, Hochedlinger K. Cell Stem Cell. 2017 May 4;20(5):706-719.e7. doi: 10.1016/j.stem.2017.03.002. Epub 2017 Mar 30. PMID 28366588

Plain-language summary We identified DUSP9 as a gene that explains why female stem cells have different DNA methylation levels than male cells.

Tags: DUSP9 X Chromosome Sex Differences Epigenetics Pluripotent Stem Cells DNA Methylation

Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. However, male and female blastocysts are similarly hypomethylated, indicating that sex- specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.

2015

A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs.
Choi J, Lee S, Mallard W, Clement K, Tagliazucchi GM, Lim H, Choi IY, Ferrari F, Tsankov AM, Pop R, Lee G, Rinn JL, Meissner A, Park PJ, Hochedlinger K. Nat Biotechnol. 2015 Nov;33(11):1173-81. doi: 10.1038/nbt.3388. Epub 2015 Oct 26. PMID 26501951

Plain-language summary We demonstrated that stem cells made from adult skin are molecularly and functionally equivalent to embryonic stem cells.

Tags: Epigenetic Memory Stem Cell Equivalence Epigenetics Gene Expression Reprogramming iPSCs vs ESCs

The equivalence of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) remains controversial. Here we use genetically matched hESC and hiPSC lines to assess the contribution of cellular origin (hESC vs. hiPSC), the Sendai virus (SeV) reprogramming method and genetic background to transcriptional and DNA methylation patterns while controlling for cell line clonality and sex. We find that transcriptional and epigenetic variation originating from genetic background dominates over variation due to cellular origin or SeV infection. Moreover, the 49 differentially expressed genes we detect between genetically matched hESCs and hiPSCs neither predict functional outcome nor distinguish an independently derived, larger set of unmatched hESC and hiPSC lines. We conclude that hESCs and hiPSCs are molecularly and functionally equivalent and cannot be distinguished by a consistent gene expression signature. Our data further imply that genetic background variation is a major confounding factor for transcriptional and epigenetic comparisons of pluripotent cell lines, explaining some of the previously observed differences between genetically unmatched hESCs and hiPSCs.

Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells.
Liao J, Karnik R, Gu H, Ziller MJ, Clement K, Tsankov AM, Akopian V, Gifford CA, Donaghey J, Galonska C, Pop R, Reyon D, Tsai SQ, Mallard W, Joung JK, Rinn JL, Gnirke A, Meissner A. Nat Genet. 2015 May;47(5):469-78. doi: 10.1038/ng.3258. Epub 2015 Mar 30. PMID 25822089

Plain-language summary We disrupted DNA methylation enzymes in human stem cells to study their role in development and gene regulation.

Tags: Human ESCs Epigenetics Knockout Lines DNMTs CRISPR DNA Methylation Epigenetic Regulation

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.

Age- and pregnancy-associated DNA methylation changes in mammary epithelial cells.
Huh SJ, Clement K, Jee D, Merlini A, Choudhury S, Maruyama R, Yoo R, Chytil A, Boyle P, Ran FA, Moses HL, Barcellos-Hoff MH, Jackson-Grusby L, Meissner A, Polyak K. Stem Cell Reports. 2015 Feb 10;4(2):297-311. doi: 10.1016/j.stemcr.2014.12.009. Epub 2015 Jan 22. PMID 25619437

Plain-language summary We mapped how DNA methylation changes in mammary gland cells during aging and pregnancy.

Tags: Cell Differentiation Aging Mammary Gland Epigenetics DNA Methylation Pregnancy

Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24(+)CD61(+)CD29(hi), CD24(+)CD61(+)CD29(lo), and CD24(+)CD61(-)CD29(lo) cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFbeta signaling as key regulators of CD24(+)CD61(+)CD29(lo) cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells.

2014

Locally disordered methylation forms the basis of intratumor methylome variation in chronic lymphocytic leukemia.
Landau DA, Clement K, Ziller MJ, Boyle P, Fan J, Gu H, Stevenson K, Sougnez C, Wang L, Li S, Kotliar D, Zhang W, Ghandi M, Garraway L, Fernandes SM, Livak KJ, Gabriel S, Gnirke A, Lander ES, Brown JR, Neuberg D, Kharchenko PV, Hacohen N, Getz G, Meissner A, Wu CJ. Cancer Cell. 2014 Dec 8;26(6):813-825. doi: 10.1016/j.ccell.2014.10.012. PMID 25490447

Plain-language summary We found that disordered DNA methylation patterns are a hallmark of chronic lymphocytic leukemia and link to disease progression.

Tags: Tumor Evolution CLL Epigenetics Disordered Methylation Epigenetic Heterogeneity DNA Methylation

Intratumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of 104 primary chronic lymphocytic leukemias (CLLs). Compared with 26 normal B cell samples, CLLs consistently displayed higher intrasample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylation disorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to that of genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories.

Long-term persistence and development of induced pancreatic beta cells generated by lineage conversion of acinar cells.
Li W, Cavelti-Weder C, Zhang Y, Clement K, Donovan S, Gonzalez G, Zhu J, Stemann M, Xu K, Hashimoto T, Yamada T, Nakanishi M, Zhang Y, Zeng S, Gifford D, Meissner A, Weir G, Zhou Q. Nat Biotechnol. 2014 Dec;32(12):1223-30. doi: 10.1038/nbt.3082. Epub 2014 Nov 17. PMID 25402613

Plain-language summary We successfully converted pancreatic cells into insulin-producing beta cells in mice, which persisted and functioned for over a year.

Tags: Gene Therapy Beta Cells Diabetes In Vivo Conversion Epigenetics Pancreas Lineage Reprogramming

Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, the survival and function of lineage-reprogrammed cells in vivo over the long term has not been examined. Here, using an improved method for in vivo conversion of adult mouse pancreatic acinar cells toward beta cells, we show that induced beta cells persist for up to 13 months (the length of the experiment), form pancreatic islet-like structures and support normoglycemia in diabetic mice. Detailed molecular analyses of induced beta cells over 7 months reveal that global DNA methylation changes occur within 10 d, whereas the transcriptional network evolves over 2 months to resemble that of endogenous beta cells and remains stable thereafter. Progressive gain of beta-cell function occurs over 7 months, as measured by glucose-regulated insulin release and suppression of hyperglycemia. These studies demonstrate that lineage- reprogrammed cells persist for >1 year and undergo epigenetic, transcriptional, anatomical and functional development toward a beta-cell phenotype.

2012

Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.
Boyle P, Clement K, Gu H, Smith ZD, Ziller M, Fostel JL, Holmes L, Meldrim J, Kelley F, Gnirke A, Meissner A. Genome Biol. 2012 Oct 3;13(10):R92. doi: 10.1186/gb-2012-13-10-r92. PMID 23034176

Plain-language summary We described a faster and cheaper method for mapping DNA methylation across the genome.

Tags: Epigenomics High-Throughput Sequencing Epigenetics Methodology RRBS DNA Methylation

Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.

Epigenomics and chromatin dynamics.
Akopian V, Chan MM, Clement K, Galonska C, Gifford CA, Lehtola E, Liao J, Samavarchi-Tehrani P, Sindhu C, Smith ZD, Tsankov AM, Webster J, Zhang Y, Ziller MJ, Meissner A. Genome Biol. 2012 Feb 24;13(2):313. doi: 10.1186/gb-2012-13-2-313. PMID 22364154

Plain-language summary A report on the 2012 Keystone Symposium on Epigenomics and Chromatin Dynamics.

Tags: Epigenomics Epigenetics Gene Regulation Conference Report Structural Biology Chromatin

A report of the 'Joint Keystone Symposium on Epigenomics and Chromatin Dynamics', Keystone, Colorado, 17-22 January 2012.

2010

PathGen: a transitive gene pathway generator.
Clement K, Gustafson N, Berbert A, Carroll H, Merris C, Olsen A, Clement M, Snell Q, Allen J, Roper RJ. Bioinformatics. 2010 Feb 1;26(3):423-5. doi: 10.1093/bioinformatics/btp661. Epub 2009 Dec 4. PMID 19965882

Plain-language summary We developed PathGen, a software tool that combines data from multiple sources to visualize gene interaction pathways.

Tags: Bioinformatics Tool Data Integration Gene Networks Systems Biology Pathway Analysis

SUMMARY: Many online sources of gene interaction networks supply rich visual data regarding gene pathways that can aid in the study of biological processes, disease research and drug discovery. PathGen incorporates data from several sources to create transitive connections that span multiple gene interaction databases. Results are displayed in a comprehensible graphical format, showing gene interaction type and strength, database source and microarray expression data. These features make PathGen a valuable tool for in silico discovery of novel gene interaction pathways, which can be experimentally tested and verified. The usefulness of PathGen interaction analyses was validated using genes connected to the altered facial development related to Down syndrome. AVAILABILITY: http://dna.cs.byu.edu/pathgen. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Further information is available at http://dna.cs.byu.edu/pathgen/PathGenSupplemental.pdf.