Author Correction: Predictable and precise template-free CRISPR editing of pathogenic variants.

In this Article, a data processing error affected Fig. 3e and Extended Data Table 2; these errors have been corrected online.

Using CRISPR to understand and manipulate gene regulation.

Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.

Predictable and precise template-free CRISPR editing of pathogenic variants.

Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5-11% of Cas9 guide RNAs targeting the human genome are 'precise-50', yielding a single genotype comprising greater than or equal to 50% of all major editing products. We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky-Pudlak syndrome and Menkes disease. This study establishes an approach for precise, template-free genome editing.

Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay.

A key mechanism in cellular regulation is the ability of the transcriptional machinery to physically access DNA. Transcription factors interact with DNA to alter the accessibility of chromatin, which enables changes to gene expression during development or disease or as a response to environmental stimuli. However, the regulation of DNA accessibility via the recruitment of transcription factors is difficult to study in the context of the native genome because every genomic site is distinct in multiple ways. Here we introduce the multiplexed integrated accessibility assay (MIAA), an assay that measures chromatin accessibility of synthetic oligonucleotide sequence libraries integrated into a controlled genomic context with low native accessibility. We apply MIAA to measure the effects of sequence motifs on cell type-specific accessibility between mouse embryonic stem cells and embryonic stem cell-derived definitive endoderm cells, screening 7905 distinct DNA sequences. MIAA recapitulates differential accessibility patterns of 100-nt sequences derived from natively differential genomic regions, identifying E-box motifs common to epithelial-mesenchymal transition driver transcription factors in stem cell-specific accessible regions that become repressed in endoderm. We show that a single binding motif for a key regulatory transcription factor is sufficient to open chromatin, and classify sets of stem cell-specific, endoderm-specific, and shared accessibility-modifying transcription factor motifs. We also show that overexpression of two definitive endoderm transcription factors, T and Foxa2, results in changes to accessibility in DNA sequences containing their respective DNA-binding motifs and identify preferential motif arrangements that influence accessibility.

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells.

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.

Machine learning based CRISPR gRNA design for therapeutic exon skipping.

Restoring gene function by the induced skipping of deleterious exons has been shown to be effective for treating genetic disorders. However, many of the clinically successful therapies for exon skipping are transient oligonucleotide-based treatments that require frequent dosing. CRISPR-Cas9 based genome editing that causes exon skipping is a promising therapeutic modality that may offer permanent alleviation of genetic disease. We show that machine learning can select Cas9 guide RNAs that disrupt splice acceptors and cause the skipping of targeted exons. We experimentally measured the exon skipping frequencies of a diverse genome-integrated library of 791 splice sequences targeted by 1,063 guide RNAs in mouse embryonic stem cells. We found that our method, SkipGuide, is able to identify effective guide RNAs with a precision of 0.68 (50% threshold predicted exon skipping frequency) and 0.93 (70% threshold predicted exon skipping frequency). We anticipate that SkipGuide will be useful for selecting guide RNA candidates for evaluation of CRISPR-Cas9-mediated exon skipping therapy.

Detection of gene cis-regulatory element perturbations in single-cell transcriptomes.

We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss of target gene expression occurs in only ~5% of cells. This low rate of biallelic loss significantly increases the number of cells required to detect the consequences of changes to the regulatory genome, but can be ameliorated by transcript-targeted sequencing. Based on our experimental results we model the power to detect regulatory genome induced transcriptomic effects based on the rate of mono/biallelic loss, baseline gene expression, and the number of cells per target gRNA.

Estimating Craniofacial Growth Cessation: Comparison of Asymptote- and Rate-Based Methods.

OBJECTIVE: To identify differences between asymptote- and rate-based methods for estimating age and size at growth cessation in linear craniofacial measurements. DESIGN: This is a retrospective, longitudinal study. Five linear measurements were collected from lateral cephalograms as part of the Craniofacial Growth Consortium Study (CGCS). Four estimates of growth cessation, including 2 asymptote- (GCasym, GCerr) and 2 rate-based (GCabs, GC10%) methods, from double logistic models of craniofacial growth were compared. PARTICIPANTS: Cephalometric data from participants in 6 historic longitudinal growth studies were included in the CGCS. At least 1749 individuals (870 females, 879 males), unaffected by craniofacial anomalies, were included in all analyses. Individuals were represented by a median of 11 images between 2.5 and 31.3 years of age. RESULTS: GCasym consistently occurred before GCerr and GCabs consistently occurred before GC10% within the rate-based approaches. The ordering of the asymptote-based methods compared to the rate-based methods was not consistent across measurements or between males and females. Across the 5 measurements, age at growth cessation ranged from 13.56 (females, nasion-basion, GCasym) to 24.39 (males, sella-gonion, GCerr). CONCLUSIONS: Adolescent growth cessation is an important milestone for treatment planning. Based on our findings, we recommend careful consideration of specific definitions of growth cessation in both clinical and research settings since the most appropriate estimation method may differ according to patients' needs. The different methods presented here provide useful estimates of growth cessation that can be applied to raw data and to a variety of statistical models of craniofacial growth.

Geometric morphometric analysis of growth patterns among facial types.

INTRODUCTION: Extreme patterns of vertical facial divergence are of great importance to clinicians because of their association with dental malocclusion and functional problems of the orofacial complex. Understanding the growth patterns associated with vertical facial divergence is critical for clinicians to provide optimal treatment. This study evaluates and compares growth patterns from childhood to adulthood among 3 classifications of vertical facial divergence using longitudinal, lateral cephalograms from the Craniofacial Growth Consortium Study. METHODS: Participants (183 females, 188 males) were classified into 1 of 3 facial types on the basis of their adult mandibular plane angle (MPA): hyperdivergent (MPA >39°; n = 40), normodivergent (28° ≤ MPA ≤ 39°; n = 216), and hypodivergent (MPA <28°; n = 115). Each individual had 5 cephalograms between ages 6 and 20 years. A set of 36 cephalometric landmarks were digitized on each cephalogram. Landmark configurations were superimposed to align 5 homologous landmarks of the anterior cranial base and scaled to unit centroid size. Growth trajectories were calculated using multivariate regression for each facial type and sex combination. RESULTS: Divergent growth trajectories were identified among facial types, finding more similarities in normodivergent and hypodivergent growth patterns than either share with the hyperdivergent group. Through the use of geometric morphometric methods, new patterns of facial growth related to vertical facial divergence were identified. Hyperdivergent growth exhibits a downward rotation of the maxillomandibular complex relative to the anterior cranial base, in addition to the increased relative growth of the lower anterior face. Conversely, normodivergent and hypodivergent groups exhibit stable positioning of the maxilla relative to the anterior cranial base, with the forward rotation of the mandible. Furthermore, the hyperdivergent maxilla and mandible become relatively shorter and posteriorly positioned with age compared with the other groups. CONCLUSIONS: This study demonstrates how hyperdivergent growth, particularly restricted growth and positioning of the maxilla, results in a higher potential risk for Class II malocclusion. Future work will investigate growth patterns within each classification of facial divergence.

Clinical implications of age-related change of the mandibular plane angle.

OBJECTIVE: To identify trajectories of ontogenetic change in the mandibular plane angle (MPA) and to describe the influence of sex and other factors on MPA during growth. SETTING/SAMPLE: The data consisted of 7026 MPA measurements from lateral cephalographs representing longitudinal series from ages 6 to 21 for 728 individuals from the Craniofacial Growth Consortium Study (CGCS). MATERIALS AND METHODS: Facial type was determined from MPA for each assessment, with the assessment closest to age 18 representing the adult facial type. The sample includes 366 males and 362 females, each with between 2 and 15 cephalographs. The mean number of cephalographs per individual is 10. Variation in childhood MPA (earliest assessment between 6 and 9 years of age) and adult MPA (closest assessment to age 18 between 15 and 21 years of age), and change in MPA from childhood to adulthood were compared by sex and adult facial type using ANOVA and post hoc t tests. RESULTS: Mandibular plane angle decreased from childhood to adulthood in 92% of males and 81% of females, yet increased in 36% of males and 50% of females with the hyper-divergent adult facial type. Childhood MPA and overall change in MPA were significantly different by adult facial type. CONCLUSIONS: Adult facial type is associated with differences in childhood MPA and change in MPA during growth. There are multiple ontogenetic pathways by which an individual can achieve a normo-divergent adult facial type, and an individual's childhood MPA does not necessarily correspond to his or her adult facial type.

A synergistic DNA logic predicts genome-wide chromatin accessibility.

Enhancers and promoters commonly occur in accessible chromatin characterized by depleted nucleosome contact; however, it is unclear how chromatin accessibility is governed. We show that log-additive cis-acting DNA sequence features can predict chromatin accessibility at high spatial resolution. We develop a new type of high-dimensional machine learning model, the Synergistic Chromatin Model (SCM), which when trained with DNase-seq data for a cell type is capable of predicting expected read counts of genome-wide chromatin accessibility at every base from DNA sequence alone, with the highest accuracy at hypersensitive sites shared across cell types. We confirm that a SCM accurately predicts chromatin accessibility for thousands of synthetic DNA sequences using a novel CRISPR-based method of highly efficient site-specific DNA library integration. SCMs are directly interpretable and reveal that a logic based on local, nonspecific synergistic effects, largely among pioneer TFs, is sufficient to predict a large fraction of cellular chromatin accessibility in a wide variety of cell types.

Predicting adult facial type from mandibular landmark data at young ages.

OBJECTIVES: To assess the potential of predicting adult facial types at different stages of mandibular development. SETTING AND SAMPLE POPULATION: A total of 941 participants from the Bolton-Brush, Denver, Fels, Iowa, Michigan and Oregon growth studies with longitudinal lateral cephalograms (total of 7166) between ages 6-21 years. MATERIAL AND METHODS: Each participant was placed into one of three facial types based on mandibular plane angle (MPA) from cephalograms taken closest to 18 years of age (range of 15-21 years): hypo-divergent (MPA < 28°), normo-divergent (28°≤ MPA ≤ 39°) and hyper-divergent (MPA > 39°). Cephalograms were categorized into 13 age groups 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18-21. Twenty-three two-dimensional anatomical landmarks were digitized on the mandible and superimposed using generalized Procrustes analysis, which projects landmarks into a common shape space. Data were analysed within age categories using stepwise discriminant analysis to identify landmarks that distinguish adult facial types and by jackknife cross-validation to test how well young individuals can be reclassified into their adult facial types. RESULTS: Although each category has multiple best discriminating landmarks among adult types, three landmarks were common across nearly all age categories: menton, gonion and articulare. Individuals were correctly classified better than chance, even among the youngest age category. Cross-validation rates improved with age, and hyper- and hypo-divergent groups have better reclassification rates than the normo-divergent group. CONCLUSIONS: The discovery of important indicators of adult facial type in the developing mandible helps improve our capacity to predict adult facial types at a younger age.

A distant trophoblast-specific enhancer controls HLA-G expression at the maternal-fetal interface.

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal-fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.

Early Maturity as the New Normal: A Century-long Study of Bone Age.

BACKGROUND: Epiphyseal fusion (EF) marks the completion of longitudinal bone growth, a critical milestone monitored during treatment of skeletal growth and/or developmental disorders. Recently, a trend toward accelerated skeletal maturation in children has been documented. Because current methods for assessing skeletal maturation include children in their reference populations born as early as the 1930s, the timing of EF events in contemporary patients may differ substantially from those standards. QUESTIONS/PURPOSES: (1) Do children today initiate the process of EF in the hand and wrist earlier than past generations on which maturity standards are based? (2) Do children today complete EF in the hand and wrist earlier than past generations on which maturity standards are based? METHODS: A total of 1292 children (665 males, 627 females) participating in the Fels Longitudinal Study, born between 1915 and 2006, were included in this retrospective, observational study. Each participant had between one and 39 serial left hand-wrist radiographs during childhood obtained specifically for research purposes. Main outcomes were the chronological age at the first sign of EF initiation (EF-I) and the first chronological age when EF was complete (EF-C) in the radius and ulna, and metacarpals and phalanges of the first, third, and fifth rays according to criteria of the Fels method. EF is a reliable metric with an average κ agreement statistic of 0.91. Penalized B-splines were used to model the changes in EF-I and EF-C ages and to identify changes across continuous birth years with major comparisons between children born in 1935 and 1995. RESULTS: Approximately half of the epiphyses of the hand and wrist examined exhibited earlier EF-I and/or earlier EF-C in children born in 1995 compared with those born in 1935. The age at each milestone (EF-I and EF-C) decreased by as much as 6.7 and 6.8 months in males and 9.8 and 9.7 months in females, respectively. This change occurred gradually over the past century. The more proximal traits (EF of the distal radius, distal ulna, and metacarpals) were more likely to experience a shift in timing, whereas timing of EF in the phalanges remained relatively stable across birth years. CONCLUSIONS: A trend has occurred over the past century in the timing of EF, in both initiation and completion of the process, for many of the bones of the hand and wrist. Earlier EF reflects modern population advances in both skeletal and sexual maturation. Shifts in the timing of EF have the potential to influence treatment strategies for skeletal growth and/or developmental disorders such as scoliosis or leg length inequality, moving treatment windows to earlier ages. Earlier EF-I and EF-C identified in this study signals a need to reevaluate the timing of maturational milestones and current standards for skeletal assessment. LEVEL OF EVIDENCE: Level II, prognostic study.

Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal.

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.

Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation.

Embryonic stem (ES) cells hold great promise with respect to their potential to be differentiated into desired cell types. Of interest are organs derived from the definitive endoderm, such as the pancreas and liver, and animal studies have revealed an essential role for Nodal in development of the definitive endoderm. Activin A is a related TGFß member that acts through many of the same downstream signaling effectors as Nodal and is thought to mimic Nodal activity. Detailed characterization of ES cell-derived endodermal cell types by gene expression analysis in vitro and functional analysis in vivo reveal that, despite their similarity in gene expression, Nodal and Activin-derived endodermal cells exhibit a distinct difference in functional competence following transplantation into the developing mouse embryo. Pdx1-expressing cells arising from the respective endoderm populations exhibit extended differences in their competence to mature into insulin/c-peptide-expressing cells in vivo. Our findings underscore the importance of functional cell-type evaluation during stepwise differentiation of stem cells.

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states.

The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.

An integrated model of multiple-condition ChIP-Seq data reveals predeterminants of Cdx2 binding.

Regulatory proteins can bind to different sets of genomic targets in various cell types or conditions. To reliably characterize such condition-specific regulatory binding we introduce MultiGPS, an integrated machine learning approach for the analysis of multiple related ChIP-seq experiments. MultiGPS is based on a generalized Expectation Maximization framework that shares information across multiple experiments for binding event discovery. We demonstrate that our framework enables the simultaneous modeling of sparse condition-specific binding changes, sequence dependence, and replicate-specific noise sources. MultiGPS encourages consistency in reported binding event locations across multiple-condition ChIP-seq datasets and provides accurate estimation of ChIP enrichment levels at each event. MultiGPS's multi-experiment modeling approach thus provides a reliable platform for detecting differential binding enrichment across experimental conditions. We demonstrate the advantages of MultiGPS with an analysis of Cdx2 binding in three distinct developmental contexts. By accurately characterizing condition-specific Cdx2 binding, MultiGPS enables novel insight into the mechanistic basis of Cdx2 site selectivity. Specifically, the condition-specific Cdx2 sites characterized by MultiGPS are highly associated with pre-existing genomic context, suggesting that such sites are pre-determined by cell-specific regulatory architecture. However, MultiGPS-defined condition-independent sites are not predicted by pre-existing regulatory signals, suggesting that Cdx2 can bind to a subset of locations regardless of genomic environment. A summary of this paper appears in the proceedings of the RECOMB 2014 conference, April 2-5.

A multi-parametric flow cytometric assay to analyze DNA-protein interactions.

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA-protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein-DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro.

Do Secular Trends in Skeletal Maturity Occur Equally in Both Sexes?

BACKGROUND: Skeletal maturity assessment provides information on a child's physical development and expectations based on chronological age. Given recently recognized trends for earlier maturity in a variety of systems, most notably puberty, examination of sex-specific secular trends in skeletal maturation is important. For the orthopaedist, recent trends and changes in developmental timing can affect clinical management (eg, treatment timing) if they are currently based on outdated sources. QUESTIONS/PURPOSES: (1) Has the male or female pediatric skeleton experienced a secular trend for earlier maturation over the past 80 years? (2) Do all indicators of maturity trend in the same direction (earlier versus later)? METHODS: In this retrospective study, a total of 1240 children were examined longitudinally through hand-wrist radiographs for skeletal maturity based on the Fels method. All subjects participate in the Fels Longitudinal Study based in Ohio and were born between 1930 and 1964 for the "early" cohort and between 1965 and 2001 for the "recent" cohort. Sex-specific secular trends were estimated for (1) mean relative skeletal maturity through linear mixed models; and (2) median age of maturation for individual maturity indicators through logistic regression and generalized estimating equations. RESULTS: Overall relative skeletal maturity was significantly advanced in the recent cohort (maximum difference of 5 months at age 13 years for girls, 4 months at age 15 years for boys). For individual maturity indicators, the direction and magnitude of secular trends varied by indicator type and sex. The following statistically significant secular trends were found: (1) earlier maturation of indicators of fusion in both sexes (4 months for girls, 3 months for boys); (2) later maturation of indicators of projection in long bones in both sexes (3 months for girls, 2 months for boys); (3) earlier maturation of indicators of density (4 months) and projection (3 months) in carpals and density in long bones (6 months), for girls only; and (4) later maturation of indicators of long bone shape (3 months) for boys only. CONCLUSIONS: A secular trend has occurred in the tempo of maturation of individual components of the pediatric skeleton, and it has occurred in a sex-specific manner. The mosaic nature of this trend, with both earlier and later maturation of individual components of the skeletal age phenotype, calls for greater attention to specific aspects of maturation in addition to the overall skeletal age estimate. The Fels method is currently the most robust method for capturing these components, and future work by our group will deliver an updated, user-friendly version of the Fels assessment tool. CLINICAL RELEVANCE: Appreciation of sex-specific secular changes in maturation is important for clinical management, including treatment timing, of orthopaedic patients, because children today exhibit a different pattern of maturation than children on whom original maturity assessments were based (including Fels and Greulich-Pyle).