Neurodevelopmental Disorders Research Articles

Involvement of extracellular vesicle microRNA clusters in developing healthy and Rett syndrome brain organoids

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions, no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0, day 13, day 40, and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover, the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together, our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment, displaying promising prospects as biomarkers for monitoring RTT progression.Graphical

Defining Developmental Regression in Rare Neurodevelopmental Disorders of Genetic Etiology: A Scoping Review

Background: Some genetic neurodevelopmental disorders (NDDs) are linked to a loss of acquired abilities. No universal term or severity measure exists for this phenomenon. This scoping review aims further to define developmental regression in NDDs of genetic etiology. Method: We used the PRISMA checklist and searched PubMed, medRxiv, and Google Scholar for developmental regression literature. After data extraction, qualitative (e.g., assessment methods) and quantitative (e.g., mentioned NDDs) data were analyzed. Results: A total of 59 relevant articles from 2074 unique records were identified, associating 18 NDDs of genetic etiology with developmental regression. Multiple terms (e.g., loss of skills, deterioration) and definitions were used across syndromes. Conclusions: A uniform definition of developmental regression was formulated based on literature diversity and NDD heterogeneity. The study also offers guidance on identifying and monitoring developmental regression and its underlying causes.

Evaluating the association between placenta DNA methylation and cognitive functions in the offspring

The placenta plays a crucial role in protecting the fetus from environmental harm and supports the development of its brain. In fact, compromised placental function could predispose an individual to neurodevelopmental disorders. Placental epigenetic modifications, including DNA methylation, could be considered a proxy of placental function and thus plausible mediators of the association between intrauterine environmental exposures and genetics, and childhood and adult mental health. Although neurodevelopmental disorders such as autism spectrum disorder have been investigated in relation to placenta DNA methylation, no studies have addressed the association between placenta DNA methylation and child’s cognitive functions. Thus, our goal here was to investigate whether the placental DNA methylation profile measured using the Illumina EPIC array is associated with three different cognitive domains (namely verbal score, perceptive performance score, and general cognitive score) assessed by the McCarthy Scales of Children’s functions in childhood at age 4. To this end, we conducted epigenome-wide association analyses, including data from 255 mother-child pairs within the INMA project, and performed a follow-up functional analysis to help the interpretation of the findings. After multiple-testing correction, we found that methylation at 4 CpGs (cg1548200, cg02986379, cg00866476, and cg14113931) was significantly associated with the general cognitive score, and 2 distinct differentially methylated regions (DMRs) (including 27 CpGs) were significantly associated with each cognitive dimension. Interestingly, the genes annotated to these CpGs, such as DAB2, CEP76, PSMG2, or MECOM, are involved in placenta, fetal, and brain development. Moreover, functional enrichment analyses of suggestive CpGs (p < 1 × 10−4) revealed gene sets involved in placenta development, fetus formation, and brain growth. These findings suggest that placental DNA methylation could be a mechanism contributing to the alteration of important pathways in the placenta that have a consequence on the offspring’s brain development and cognitive function.

Correction to “Role of mitochondria‐endoplasmic reticulum contacts in neurodegenerative, neurodevelopmental and neuropsychiatric conditions”

Correction to “Role of mitochondria‐endoplasmic reticulum contacts in neurodegenerative, neurodevelopmental and neuropsychiatric conditions”

Autism-Related Cc2d1a Heterozygous Mice: Increased Levels of miRNAs Retained in DNA/RNA Hybrid Profiles (R-Loop)

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a highly variable expression of phenotypes (restricted interest or activity and repetitive behavior in communication and social interactions), genes (mutation), markers (alteration of transcription) and pathways. Loss of function of the CC2D1A gene appears to primarily affect the brain, leading to a range of behavioral problems in humans. In our study published in 2020, we found that the expressions of miR-19a-3p, miR-361-5p, miR-150-5p, miR-3613-3p, miR-126-3p and miR-499a-5p were downregulated in the serum samples of autistic patients, their families and mouse models (Cc2d1a +/− and valproic acid treated males). Here, acquired non-Mendelian hereditary character in a genetically defined mouse model of autism (Cc2d1a +/−) correlates with the transcriptional alteration of five miRNAs. We seek to test the hypothesis that miRNA levels vary by changes in RNA/DNA structure during development, thereby creating transcription alteration and cell memory. Behavioral tests were conducted on the offspring of Cc2d1a (+/−) mutant and control mice, such as novel object, social interaction, marble burying and tail suspension behavior. Two RNA fractions were isolated from mouse hippocampal tissues and sperm cells via standard TRIzol extraction: free RNA and the fraction of RNA bound to DNA in the form of a DNA/RNA hybrid (R-loop). The expression levels of miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p and miR-499a-5p were investigated by quantitative real-time RT-PCR. We report differences in the distribution of five miRNAs in the hippocampus between male and female mice, particularly in colonies of Cc2d1a (+/−) mice. Furthermore, the number of miRNAs engaged in the DNA/RNA hybrid fraction is generally higher in the mutant pedigree than in the control group. On the other hand, in sperm, both fractions are at lower levels than in controls. R-loops contribute to the physiology and pathology of organisms including human disease. Here, we report a variation in five miRNA levels between gender and tissue. Our results suggest that the transcription levels of these five miRNAs are directly regulated by their RNA.

Combining optical genome mapping and RNA-seq for structural variants detection and interpretation in unsolved neurodevelopmental disorders

BackgroundStructural variations (SVs) are key genetic contributors to neurodevelopmental disorders (NDDs). Exome sequencing (ES), the current first-line tool for genetic testing of NDDs, falls short in SVs detection. This diagnostic gap is being actively addressed by new methods such as optical genome mapping (OGM).MethodsThis study evaluated the utility of combining OGM and RNA-seq in the detection and interpretation of SVs in ES-negative NDDs. OGM was performed in 43 patients with NDDs with inconclusive ES results. Candidate SVs were selected based on disease association and pathogenicity evaluation, and further validated or reconstructed by alternative methods, including long-read sequencing for a complex rearrangement event. RNA-Seq was performed on blood samples from patients with candidate SVs to facilitate interpretation of pathogenicity.ResultsOGM detected four candidate SVs, and RNA-seq confirmed the pathogenicity of three SVs in the patient cohort. This combined approach solved three cases—two cases with de novo SVs in genes associated with autosomal dominant NDDs, including a deletion encompassing the promoter and 5′UTR of MBD5 and an intragenic duplication of PAFAH1B1, and a third case possessing an intragenic duplication in trans with a pathogenic single-nucleotide variant of PLA2G6, associated with autosomal recessive NDDs. The expression alteration of the affected genes and the tandem positioning of two intragenic duplications were confirmed by RNA-seq. In the fourth case, OGM detected a complex rearrangement involving chromosomes 2 and 6, much more complex than the de novo t(2:6)(q13;q15) indicated by conventional cytogenetic analysis. Reconstruction showed that 17 segments of 6q15 spanning 9.3 Mb were disarranged and joined 2q11.2, with four breakpoints detected in the 5′ and 3′ non-coding region of the NDD-associated gene SYNCRIP. RNA-seq revealed largely preserved SYNCRIP expression, leaving the pathogenicity of this complex rearrangement event uncertain.ConclusionsSVs in ES-negative NDDs can be identified by OGM, which is particularly useful for SVs in non-coding regions not covered by ES. OGM helps to construct complex SVs and provides information on the location and orientation of duplications, which is crucial for pathogenicity interpretation. The integration of RNA-seq facilitates the interpretation of the functional consequences of SVs at the transcriptional level. These findings demonstrate the utility and feasibility of combining OGM and RNA-seq in ES-negative cases with NDDs.

Care bundle for preventing intraventricular hemorrhage in premature infants: a best practice implementation project.

Intraventricular hemorrhages remain a major problem in neonatology, because their complications affect neonatal morbidity, mortality, and long-term neurodevelopmental outcomes. The aim of this project was to prevent intraventricular hemorrhage in premature infants during their first days of life in a neonatal intensive care unit (NICU). This pre- and post-implementation clinical audit project used the JBI Evidence Implementation Framework and was conducted in a tertiary-level Spanish NICU with a consecutive sample. A baseline audit was conducted using 13 audit criteria derived from JBI summaries of the best available evidence. This was followed by the implementation of an action plan, which included a care bundle and health care professionals' education. These improvement strategies were then evaluated using a follow-up audit. The baseline and follow-up audits evaluated 54 and 56 infants, respectively. The follow-up audit showed 100% improvement for Criteria 2, 3, 6, and 7, which covered sleep safety and noise. Criteria 12 and 13, which covered cord clamping and positioning the infant, improved by 25.99%. Criterion 9, on antenatal corticosteroids, showed a slight improvement of 5.56%. This study increased compliance with an evidence-based, family-centered care approach to preventing intraventricular hemorrhage in premature infants. This was achieved by conducting a baseline and follow-up audit, implementing a training program, and keeping more comprehensive nursing records. Further studies could assess the long-term effectiveness of interventions and/or the incidence of intraventricular hemorrhage and neurodevelopmental disorders in premature infants. http://links.lww.com/IJEBH/A262.

Research progress of the relationship between phosphoprotein phosphatases (PPPs) and neurodevelopmental disorders

Research progress of the relationship between phosphoprotein phosphatases (<scp>PPPs</scp>) and neurodevelopmental disorders

Long-read genome sequencing and variant reanalysis increase diagnostic yield in neurodevelopmental disorders

Variant detection from long-read genome sequencing (lrGS) has proven to be more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences HiFi technology on 96 short-read-negative probands with rare diseases that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, nine of which (8/96, ~9.4%) harbored pathogenic or likely pathogenic variants. Nine probands (~9.4%) had variants that were accurately called in both srGS and lrGS and represent changes to clinical interpretation, mostly from recently published gene-disease associations. Seven cases included variants that were only correctly interpreted in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older srGS data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

Neurodevelopmental Impairments in Adult Psychosomatic Patients

Background/Objectives: Neuropsychological, neurodevelopmental, or minimal cerebral dysfunctions (MCD) can be found in many patients with mental disorders. They can be masked by other symptoms, impair the course of the illness, and impair work and social participation. Despite a long history of research, there is still a lack of data on the spectrum, prevalence, and consequences of these dysfunctions in patients with chronic illness. In this study, we compared patients with and without a history of neurocognitive problems in childhood for present neuropsychological dysfunctions. Methods: A convenience sample of 1453 psychosomatic inpatients completed the MCD scale, assessing neurodevelopmental issues in childhood and current neuropsychological dysfunctions. Additional assessments were the Attention Deficit Hyperactivity Self Rating Scale (ADHS-SB) and the Symptom Checklist 90 (SCL-90). Results: Significant early neurodevelopmental problems were reported by 8.87% of the patients. This group also reported a significantly higher rate of MCD symptoms and general psychosomatic symptoms (SCL-90) as compared with other patients. Conclusions: There is a notable prevalence of neuropsychological dysfunctions in psychosomatic patients in general, and especially in those with early neurodevelopmental problems. To adequately address specific potentially participation-relevant impairments, a broader diagnostic approach is necessary, including exploration of MCD history and present neuropsychological dysfunctions.

Lipophilic compounds restore function to neurodevelopmental-associated KCNQ3 mutations

A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss– and gain–of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel’s pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.

Paternal exposure to antiseizure medications and offspring outcomes: a systematic review

BackgroundConcerns have recently been raised about risks to the fetus resulting from paternal exposure to antiseizure medications (ASMs). To address these concerns, we conducted a systematic review of the literature...

Astrocyte allocation during brain development is controlled by Tcf4-mediated fate restriction

AbstractAstrocytes in the brain exhibit regional heterogeneity contributing to regional circuits involved in higher-order brain functions, yet the mechanisms controlling their distribution remain unclear. Here, we show that the precise allocation of astrocytes to specific brain regions during development is achieved through transcription factor 4 (Tcf4)-mediated fate restriction based on their embryonic origin. Loss of Tcf4 in ventral telencephalic neural progenitor cells alters the fate of oligodendrocyte precursor cells to transient intermediate astrocyte precursor cells, resulting in mislocalized astrocytes in the dorsal neocortex. These ectopic astrocytes engage with neocortical neurons and acquire features reminiscent of dorsal neocortical astrocytes. Furthermore, Tcf4 functions as a suppressor of astrocyte fate during the differentiation of oligodendrocyte precursor cells derived from the ventral telencephalon, thereby restricting the fate to the oligodendrocyte lineage in the dorsal neocortex. Together, our findings highlight a previously unappreciated role for Tcf4 in regulating astrocyte allocation, offering additional insights into the mechanisms underlying neurodevelopmental disorders linked to Tcf4 mutations.

Case report: A novel FBXW7 gene variant causes global developmental delay

Objective: To investigate a case of neurodevelopmental disorder caused by mutation of FBXW7.Methods: Clinical data were collected from the patient, trio-WES (whole-exome sequencing) was performed on the patient and his parents (trio), and the results were verified by Sanger sequencing. RESULTS: The patient was a 2-year and 1-month old male who presented with facial dysmorphism (prominent forehead, ocular hypertelorism, and low nasal bridge), global developmental delay, language impairment, hypertonia, labial hemangioma, hydrocele, and overgrowth. The trio-WES confirmed that the child had a pathogenic de novo FBXW7 gene variant, c.1612C&amp;gt;T (p.G1n538*), a heretofore unreported locus.Conclusion: This case of developmental delay, hypotonia, and impaired language (OMIM: #620012) related to a mutation in FBXW7, is a rare genetic disorder, newly identified in recent years, and seldom reported. The presence of hypertonia, labial hemangioma, and hydrocele in this child suggests significant phenotypic heterogeneity of the disease, and the discovery of new mutant loci enriches the spectrum of pathogenic variants of the disease.

A Preliminary Investigation of Within-Word Silent Intervals Produced by Children With and Without Neurodevelopmental Disorders.

The categorization of silent intervals during speech production is necessary for accurate measurement of articulation rate and pauses. The primary purpose of this preliminary study was to examine the within-word silent interval associated with the stop closure in word-final stop consonants produced by children with and without neurodevelopmental disorders. Seven children diagnosed with either cerebral palsy or Down syndrome (i.e., children with neurodevelopmental disorders) and eight typically developing children produced a reading passage. Participants were between the ages of 11 and 16 years. Fifty-eight words from the reading passage were identified as having word-final stop consonants. The closure duration of the word-final stop consonant was calculated, both in absolute duration and percent pause time. The articulation rate of the entire passage was calculated. The number of closure durations that met or exceeded the minimum duration threshold to be considered a pause (150 ms) was examined descriptively. Children with neurodevelopmental disorders produced significantly longer closure durations and significantly slower articulation rates than typically developing children. Children with neurodevelopmental disorders produced closure durations that met or exceeded the minimum duration threshold of a pause, but typically developing children, generally, did not. These data indicate the need to examine the location of silent intervals that meet the minimum duration threshold of a pause and correct for articulatory events during the measurement of articulation rate and pauses in children with neurodevelopmental disorders.

Cellulose acetate electrophoretic separation of serum and urine proteins in Nigerian children with autism spectrum disorders

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) characterized by social communication challenges and restricted, repetitive behaviors. While genetic and environmental factors are known to contribute to ASD, the role of the immune system remains unclear. This study investigated the separation patterns of serum and urine proteins in Nigerian children with ASD compared to typically developing children and children with other NDDs.MethodsForty-seven participants aged 3–8 years were recruited, including 16 children diagnosed with ASD and 16 children with other NDDs, both according to DSM-5 criteria, along with 15 neurotypical children. Blood and urine samples were collected for protein analysis. Total protein and albumin levels were measured in both serum and urine using established methods. Protein separation in serum and urine was performed using cellulose acetate electrophoresis, followed by densitometry analysis of the electrophoretic patterns.ResultsThe results revealed no significant differences in total serum protein levels and most protein fractions between the groups. However, children with other NDDs exhibited significantly lower levels of alpha-2 globulin compared to neurotypical children. Conversely, both ASD and NDD groups showed significantly higher gamma globulin levels compared to the control group. Interestingly, spot urine protein levels were significantly higher in children with ASD compared to neurotypical children.ConclusionThe observed changes in alpha-2 and gamma globulin levels suggest potential immune system involvement in ASD and other NDDs. The higher urine protein excretion in the ASD group warrants further investigation to explore the potential of urinary protein biomarkers for ASD diagnosis.

Understanding levels of engagement and readiness for change in an eHealth sleep program for children with neurodevelopmental disorders

BackgroundChildren with neurodevelopmental disorders (NDD) experience high rates of sleep problems. The Better Nights, Better Days for Children with Neurodevelopmental DisordersTM(BNBD-NDDTM) program is an online intervention for parents of children with NDD who have insomnia/insomnia symptoms. The program has recently undergone a national implementation study (recruitment completed; data collection and analysis ongoing), where parental adherence and engagement are being evaluated. Preliminary results have shown that despite high levels of recruitment, there is less utilization of the program than the research team expected. Parental engagement may have been impacted by participants' motivation and readiness for change, as well as indirectly by the COVID-19 pandemic. The objective of the current study is to better understand engagement with the BNBD-NDDTM program concerning parental motivation and readiness for change, while considering the possible impacts of COVID-19.MethodsParents of children with NDD (n = 18) who were enrolled in the BNBD-NDDTM program for a minimum of 4 months completed exit interviews using a researcher-generated, semi-structured interview guide. During the interview, participants were asked about their engagement in the program, perspectives on their own readiness for changing their children's sleep, and the impact of COVID-19 on their engagement. Data were analyzed following an inductive content analysis approach.ResultsSeveral categories of data were generated that explain levels of engagement, including: (1) severity of sleep problems; (2) motivation for change; (3) previous strategies for sleep; (4) confidence in the program; (5) sacrifices made to change sleep; (6) maintenance of change; (7) experience with levels of support provided; and (8) impact of the COVID-19 pandemic.ConclusionParents identified several factors related to their readiness for change as contributors to their engagement level in the BNBD-NDDTM program. The COVID-19 pandemic had varied impacts on engagement for participants in this sample. Understanding parents' engagement levels within the BNBD-NDDTM eHealth program related to their motivation and readiness for change is crucial to optimize uptake and adherence to the program, improve the program's implementation and sustainability, and continue to help children with NDD to sleep better.

Prospective associations between early childhood mental health concerns and formal diagnosis of neurodevelopmental disorders in adolescence

IntroductionUnderstanding associations between psychosocial development in early childhood and formal diagnosis of neurodevelopmental disorders (NDDs) in adolescence is critical for early identification and for tailoring interventions and support. This study investigated whether the Strengths and Difficulties Questionnaire (SDQ) scores in early childhood (4-5 years) predict mental health (MH) problems as evidenced by SDQ scores and formal diagnosis of NDDs in adolescence (16-17 years).MethodsThis study analysed data from a sample of 4968 children and adolescents using data from the Longitudinal Study of Australian Children. We used hierarchical regression models to determine the association between SDQ subscales and total scores at ages 4-5 years (primary exposure) and total SDQ scores and NDD diagnoses at ages 16-17 years (outcomes) whilst controlling for sociodemographic risk factors.ResultsEach unit increase in SDQ score at age 4-5 led to a rise in SDQ scores at age 16-17. Autism and ADHD diagnoses, female gender, lower maternal education, and financial hardship were associated with higher SDQ scores at age 16-17. Furthermore, parent reported SDQ at age 4-5 was linked to higher likelihoods of formal diagnoses of ADHD, autism, and ADHD/autism at age 16-17. Additionally, social determinants of health such as female gender, culturally and linguistically diverse (CALD) backgrounds, and financial hardship were associated with increased odds of ADHD, autism, and ADHD/autism diagnoses at age 16-17.ConclusionOur findings highlight the opportunity for early identification of transdiagnostic developmental and MH issues in the preschool period. Findings also emphasise the critical role of social determinants of health in the longitudinal trajectory of MH and NDDs and highlight the need for implementing early supports for improving peer relations and behavioural support strategies. If coupled with wrap around social care, early support strategies can enhance MH and wellbeing in adolescence and beyond.

Identification of novel genetic loci related to dromedary camel (Camelus dromedarius) morphometrics, biomechanics, and behavior by genome-wide association studies

In the realm of animal breeding for sustainability, domestic camels have traditionally been valued for their milk and meat production. However, key aspects such as zoometrics, biomechanics, and behavior have often been overlooked in terms of their genetic foundations. Recognizing this gap, the present study perfomed genome-wide association analyses to identify genetic markers associated with zoometrics-, biomechanics-, and behavior-related traits in dromedary camels (Camelus dromedarius). 16 and 108 genetic markers were significantly associated (q < 0.05) at genome and chromosome-wide levels of significance, respectively, with zoometrics- (width, length, and perimeter/girth), biomechanics- (acceleration, displacement, spatial position, and velocity), and behavior-related traits (general cognition, intelligence, and Intelligence Quotient (IQ)) in dromedaries. In most association loci, the nearest protein-coding genes are linkedto neurodevelopmental and sensory disorders. This suggests that genetic variations related to neural development and sensory perception play crucial roles in shaping a dromedary camel’s physical characteristics and behavior. In summary, this research advances our understanding of the genomic basis of essential traits in dromedary camels. Identifying specific genetic markers associated with zoometrics, biomechanics, and behavior provides valuable insights into camel domestication. Moreover, the links between these traits and genes related to neurodevelopmental and sensory disorders highlight the broader implications of domestication and modern selection on the health and welfare of dromedary camels. This knowledge could guide future breeding strategies, fostering a more holistic approach to camel husbandry and ensuring the sustainability of these animals in diverse agricultural contexts.

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.