EcCas6e-based antisense crRNA for gene repression and RNA editing in microorganisms.

Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.

Whole-genome sequencing association analysis of quantitative red blood cell phenotypes: The NHLBI TOPMed program.

Whole-genome sequencing (WGS), a powerful tool for detecting novel coding and non-coding disease-causing variants, has largely been applied to clinical diagnosis of inherited disorders. Here we leveraged WGS data in up to 62,653 ethnically diverse participants from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program and assessed statistical association of variants with seven red blood cell (RBC) quantitative traits. We discovered 14 single variant-RBC trait associations at 12 genomic loci, which have not been reported previously. Several of the RBC trait-variant associations (RPN1, ELL2, MIDN, HBB, HBA1, PIEZO1, and G6PD) were replicated in independent GWAS datasets imputed to the TOPMed reference panel. Most of these discovered variants are rare/low frequency, and several are observed disproportionately among non-European Ancestry (African, Hispanic/Latino, or East Asian) populations. We identified a 3 bp indel p.Lys2169del (g.88717175_88717177TCT[4]) (common only in the Ashkenazi Jewish population) of PIEZO1, a gene responsible for the Mendelian red cell disorder hereditary xerocytosis (MIM: 194380), associated with higher mean corpuscular hemoglobin concentration (MCHC). In stepwise conditional analysis and in gene-based rare variant aggregated association analysis, we identified several of the variants in HBB, HBA1, TMPRSS6, and G6PD that represent the carrier state for known coding, promoter, or splice site loss-of-function variants that cause inherited RBC disorders. Finally, we applied base and nuclease editing to demonstrate that the sentinel variant rs112097551 (nearest gene RPN1) acts through a cis-regulatory element that exerts long-range control of the gene RUVBL1 which is essential for hematopoiesis. Together, these results demonstrate the utility of WGS in ethnically diverse population-based samples and gene editing for expanding knowledge of the genetic architecture of quantitative hematologic traits and suggest a continuum between complex trait and Mendelian red cell disorders.

Genetic mechanisms of COVID-19 and its association with smoking and alcohol consumption.

We aimed to investigate the genetic mechanisms associated with coronavirus disease of 2019 (COVID-19) outcomes in the host and to evaluate the possible associations between smoking and drinking behavior and three COVID-19 outcomes: severe COVID-19, hospitalized COVID-19 and COVID-19 infection. We described the genomic loci and risk genes associated with the COVID-19 outcomes, followed by functional analyses of the risk genes. Then, a summary data-based Mendelian randomization (SMR) analysis, and a transcriptome-wide association study (TWAS) were performed for the severe COVID-19 dataset. A two-sample Mendelian randomization (MR) analysis was used to evaluate the causal associations between various measures of smoking and alcohol consumption and the COVID-19 outcomes. A total of 26 protein-coding genes, enriched in chemokine binding, cytokine binding and senescence-related functions, were associated with either severe COVID-19 or hospitalized COVID-19. The SMR and the TWAS analyses highlighted functional implications of some GWAS hits and identified seven novel genes for severe COVID-19, including CCR5, CCR5AS, IL10RB, TAC4, RMI1 and TNFSF15, some of which are targets of approved or experimental drugs. According to our studies, increasing consumption of cigarettes per day by 1 standard deviation is related to a 2.3-fold increase in susceptibility to severe COVID-19 and a 1.6-fold increase in COVID-19-induced hospitalization. Contrarily, no significant links were found between alcohol consumption or binary smoking status and COVID-19 outcomes. Our study revealed some novel COVID-19 related genes and suggested that genetic liability to smoking may quantitatively contribute to an increased risk for a severe course of COVID-19.

Genetic predisposition to blood cell indices in relation to severe COVID-19.

Despite considerable variation in disease manifestations observed among coronavirus disease 2019 (COVID-19) patients infected with severe acute respiratory syndrome coronavirus 2, the risk factors predicting disease severity remain elusive. Recent studies suggest that peripheral blood cells play a pivotal role in COVID-19 pathogenesis. Here, we applied two-sample Mendelian randomization (MR) analyses to evaluate the potential causal contributions of blood cell indices variation to COVID-19 severity, using single-nucleotide polymorphisms (SNPs) as instrumental variables for 17 indices from the UK Biobank and INTERVAL genome-wide association studies (N = 173 480). Data on the associations between the SNPs and very severe respiratory confirmed COVID-19 were obtained from the COVID-19 host genetics initiative (N = 8779/1 001 875). We observed significant negative association between hematocrit (HCT; odds ratio, OR = 0.775, 95% confidence interval, CI = 0.635-0.915, p = 3.48E-04) or red blood cell count (OR = 0.830, 95% CI = 0.728-0.932, p = 2.19E-03) and very severe respiratory confirmed COVID-19, as well as nominal negative association of hemoglobin concentration (OR = 0.808, 95% CI = 0.673-0.943, p = 3.95E-03) with very severe respiratory confirmed COVID-19 (no effect survived multiple correction). In conclusion, the MR study supports a protective effect of high HCT and red blood cell count from very severe respiratory confirmed COVID-19, suggesting potential strategies to ameliorate/treat clinical conditions in very severe respiratory confirmed COVID-19.

Genetic Relationships between Attention-Deficit/Hyperactivity Disorder, Autism Spectrum Disorder, and Intelligence.

INTRODUCTION: Attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) commonly co-occur; both traits exert an influence on intelligence scores. Genetic relationships between these three traits are far from being clear. METHODS: The summary results of genome-wide association studies of ADHD (20,183 cases and 35,191 controls), ASD (18,381 cases and 27,969 controls), and intelligence (269,867 participants) were used for the analyses. Local genetic correlation analysis and polygenic overlap analysis were used to explore the shared genetic components between ADHD, ASD, and intelligence. Mendelian randomization (MR) analysis was used to examine the causal associations between ADHD, ASD, and intelligence. A cross-trait meta-analysis was performed to identify pleiotropic genetic variants across the three traits. RESULTS: Our results showed that intelligence has a positive and negative genetic correlation with ASD and ADHD, respectively, including three hub genomic regions showing correlated genetic effects across the three traits. Polygenic overlap analysis indicated that all the risk variants contributing to ADHD are overlapped with half of those for intelligence, and the majority of the shared variants have opposite effect directions between them. The majority of risk variants (80%) of ASD are overlapped with almost all the risk variants of intelligence (97%). Notably, some ASD/intelligence overlapping variants displayed opposing effects on these two conditions. MR analysis showed that the genetic liability to higher intelligence was associated with an increased risk for ASD (OR = 1.12) and a decreased risk for ADHD (OR = 0.78). Cross-trait meta-analyses identified 170 pleiotropic genomic loci across the three traits, including 12 novel loci. Functional analyses of the novel genes support their potential involvement in neurodevelopment. CONCLUSION: Our results suggest that ADHD is associated with inheriting a reduced set of low-intelligence alleles, whereas ASD results from incongruous effects from a mixture of high-intelligence and low-intelligence contributing alleles summed up with additional, ASD-specific risk variants not associated with intelligence.

Involvement of the long intergenic non-coding RNA LINC00461 in schizophrenia.

OBJECTIVE: LINC00461 is a highly conserved intergenic non-protein coding RNA that was implicated in schizophrenia at the genome-wide level. We aim to explore potential mechanisms underlying the involvement of LINC00461 in schizophrenia. METHODS: We performed a meta-analysis to investigate the association of LINC00461 rs410216 with schizophrenia, and evaluate the effects of the rs410216 on hippocampal volume and function using the functional magnetic resonance imaging (fMRI) analysis. We utilized the GTEx dataset to profile the expression distribution of LINC00461 across different brain regions, and to investigate the potential impact of the risk SNPs on the expression of LINC00461 and other nearby genes. We compared blood expression levels of LINC00461 between schizophrenia patients and controls. RESULTS: Here we show that single-nucleotide polymorphisms (SNPs) located in regulatory elements spanning the LINC00461 region are significantly associated with schizophrenia (index SNP rs410216, Pmeta = 1.43E-05); subjects carrying the risk allele of rs410216 showed decreased hippocampal volume. However, no significant association of the rs410216 variant with hippocampal activation was observed. Moreover, the expression level of LINC00461 mRNA was significantly lower in first-onset schizophrenia patients, and the risk allele also predicts a lower transcriptional level of LINC00461 in the hippocampus. CONCLUSION: Together, these convergent lines of evidence implicate inadequate LINC00461 expression in the hippocampus in the development of schizophrenia, providing novel insight into the genetic architecture and biological etiology of schizophrenia.

Identifying common genome-wide risk genes for major psychiatric traits.

Major psychiatric traits are genetically inter-correlated with one another, but it not well known which genes play pleiotropic effects across different traits. We curated and compared genes identified from large-scale genome-wide association studies for seven psychiatric traits, including depression, bipolar disorder, schizophrenia, autism spectrum disorder, attention-deficit/hyperactivity disorder, anxiety and neuroticism. We then explored biological functions of the top pleiotropic genes. A total of 243 cross-trait genes were identified for the seven traits. Except for autism spectrum disorder, there was significant enrichment of overlapped genes across these psychiatric traits. Chromosome 5q14.3, 11q23.2, and 7p22.3 are the three genomic regions conferring highest pleiotropic effects for these psychiatric traits. The long non-coding gene LINC00461 showed the highest pleiotropic effects on five psychiatric traits. In silico and functional studies with mice support the vital role of LINC00461 in neurodevelopment. In sum, our study provides insights into the shared genetic liability among major psychiatric traits.

Effect of the SIRT1 gene on regional cortical grey matter density in the Han Chinese population.

BACKGROUND: Our previous genome-wide association study (CONVERGE sample) identified significant association between single nucleotide polymorphisms (SNPs) near the SIRT1 gene and major depressive disorder (MDD) in Chinese populations. AIMS: To investigate whether SNPs across the SIRT1 gene locus affect regional grey matter density in the Han Chinese population. METHOD: T1-weighted structural magnetic resonance imaging was conducted on 92 healthy participants from Eastern China. Grey matter was segmented from the image, which consisted of voxel-wise grey matter density. The effect of SIRT1 SNPs on grey matter density was determined by a multiple linear regression framework. RESULTS: SNP rs4746720 was significantly associated with grey matter density in two brain cortical regions: the orbital part of the right inferior frontal gyrus and the orbital part of the left inferior frontal gyrus (family-wise error-corrected P < 0.05; voxel-wise P < 0.001). Also, rs4746720 exceeded genome-wide significance in association with MDD in our CONVERGE sample (P = 3.32 × 10-08, odds ratio 1.161). CONCLUSIONS: Our results provided evidence for a potential role of the SIRT1 gene in the brain, implying a possible pathophysiological mechanism underlying susceptibility to MDD.

Variants in TERT influencing telomere length are associated with paranoid schizophrenia risk.

Schizophrenia is one of the most severe psychiatric disorders, with a high heritability of up to 80%. Several studies have reported telomere dysfunction in schizophrenia, and common variants in the telomerase reverse transcriptase (TERT) gene. TERT is a key component of the telomerase complex that maintains telomere length by addition of telomere repeats to telomere ends, and has repeatedly shown association with mean lymphocyte telomere length (LTL). Thus, we hypothesized that TERT may be a novel susceptibility gene for schizophrenia. Using a Taqman protocol, we genotyped eight tag SNPs from the TERT locus in 1,072 patients with paranoid schizophrenia and 1,284 control subjects from a Chinese Han population. We also measured mean LTL in 98 cases and 109 controls using a quantitative PCR-based technique. Chi-square tests showed that two SNPs, rs2075786 (P = 0.0009, OR = 0.76, 95%CI = 0.65-0.90) and rs4975605 (P = 0.0026, OR = 0.73, 95%CI = 0.60-0.90), were associated with a protective effect, while rs10069690 was associated with risk of paranoid schizophrenia (P = 0.0044, OR = 1.23, 95%CI = 1.07-1.42). Additionally, the rs2736118-rs2075786 haplotype showed significant association with paranoid schizophrenia (P = 0.0013). Moreover, mean LTL correlated with rs2075786 genotypes was significantly shorter in the patient group than the control group. The present results suggest that the TERT gene may be a novel candidate involved in the development of paranoid schizophrenia.

Common variants in CACNA1C and MDD susceptibility: A comprehensive meta-analysis.

Major depressive disorder (MDD) is one of the most common psychiatric disorders with a relatively high heritability (35-40%). Though rs1006737 in the CACNA1C gene showed significant association with MDD in a British large-scale candidate association study, most of the replication analyses with relatively small sample size reported negative association. Moreover, this locus has never been identified in previous genome-wide association studies (GWAS) for MDD. Here, we conducted a comprehensive meta-analysis of the association between CACNA1C variants and MDD risk by combining all published data. Genetic data from one European GWAS and five individual follow-up studies, which include up to 12,629 patients of MDD and 28,653 controls, that is, the largest sample size on CACNA1C to date, were collected. Rs1006737 showed significant association with MDD in the fixed-effect model (Z = 2.56, P = 0.011, OR = 1.08, 95%CI = 1.04-1.12) and the association remained after reanalyzing the data according to ethnicity. We additionally analyzed other 25 SNPs, genotyped in only one replication study, across the CACNA1C locus, and found that two SNPs, rs4765905 (P = 0.041, OR = 1.05, 95%CI 1.00-1.09) and rs4765937 (P = 0.025, OR = 1.05, 95%CI 1.01-1.09) showed nominal association with MDD, while rs2239073 (P = 0.002, OR = 1.07, 95%CI 1.02-1.11) exhibited significant association with MDD, which survived from multiple corrections. Our study provides support for positive association between CACNA1C and MDD; however, the current data suggest the necessity of replication analyses in a larger-scale sample. © 2016 Wiley Periodicals, Inc.

[The application of exome sequencing in human disease].

It is estimated that approximately 85% of human disease mutations are located in protein coding regions, therefore selectively sequencing all protein coding regions (exome) would be cost-effective and an alternative strategy to identify diseases' varaints. In 2009, scientists successfully identified one missense mutation in MYH3 among 4 individuals with Freeman Sheldon syndrome (one autosomal dominant disease) through exome sequencing. Since then, exome sequencing has been widely used to identify disease causative or susceptibility genes in Mendelian disorders and complex diseases. The application of exome sequencing in human diseases were summarized in this review.

Recent advances in CRISPR-Cas9-based genome insertion technologies.

Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

PAMless SpRY exhibits a preference for the seed region for efficient targeting.

Protospacer-adjacent motif (PAM) recognition licenses Cas nucleases for genome engineering applications, thereby restricting gene targeting to PAM-containing regions. Protein engineering has led to PAM-relaxed SpCas9 variants like SpG and SpRY. Given the evolved role of PAMs in facilitating target-searching kinetics, it remains unclear how these variants quickly locate their targets. We show that SpG and SpRY exhibit a preference for the seed region. To compensate for the relaxed PAM recognition, SpRY has evolved a sequence preference for the seed region through interactions with A61R and A1322R. Furthermore, SpCas9 exhibits a significant decrease in target search kinetics on high-PAM-density DNA, slowing down up to three orders of magnitude compared to low-PAM-density DNA, suggesting the necessity for sequence recognition even in PAM-relaxed variants. This underscores the importance of considering Cas9 target-searching kinetics in SpCas9 PAMless engineering, providing valuable insights for further PAMless Cas9 protein engineering efforts.

SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution.

CRISPR mutagenesis screens conducted with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants but at a limited resolution due to the absence of protospacer adjacent motif (PAM) sequences. Here, leveraging the broad targeting scope of the near-PAMless SpRY variant, we have demonstrated that saturated SpRY mutagenesis and base editing screens can faithfully identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. We further extended this methodology to investigate a genome-wide association study (GWAS) locus at 10q22.1 associated with a red blood cell trait, where we identified potential enhancers regulating HK1 gene expression, despite not all of these enhancers exhibiting typical chromatin signatures. More importantly, our saturated base editing screens pinpoint multiple causal variants within this locus that would otherwise be missed by Bayesian statistical fine-mapping. Our approach is generally applicable to functional interrogation of all non-coding genomic elements while complementing other high-coverage CRISPR screens.

SingleQ: a comprehensive database of single-cell expression quantitative trait loci (sc-eQTLs) cross human tissues.

Mapping of expression quantitative trait loci (eQTLs) and other molecular QTLs can help characterize the modes of action of disease-associated genetic variants. However, current eQTL databases present data from bulk RNA-seq approaches, which cannot shed light on the cell type- and environment-specific regulation of disease-associated genetic variants. Here, we introduce our Single-cell eQTL Interactive Database which collects single-cell eQTL (sc-eQTL) datasets and provides online visualization of sc-eQTLs across different cell types in a user-friendly manner. Although sc-eQTL mapping is still in its early stage, our database curates the most comprehensive summary statistics of sc-eQTLs published to date. sc-eQTL studies have revolutionized our understanding of gene regulation in specific cellular contexts, and we anticipate that our database will further accelerate the research of functional genomics. Database URL: http://www.sqraolab.com/scqtl.

Bioadhesive and electroactive hydrogels for flexible bioelectronics and supercapacitors enabled by a redox-active core-shell PEDOT@PZIF-71 system.

Stretchable and conductive hydrogels are rapidly emerging as new generation candidates for wearable devices. However, the poor electroactivity and bioadhesiveness of traditional conductive hydrogels has limited their applications. Herein, a mussel-inspired strategy is proposed to prepare a specific core-shell redox-active system, consisting of a polydopamine (PDA) modified zeolitic imidazolate framework 71 (ZIF-71) core, and a poly 3,4-ethylenedioxythiopene (PEDOT) shell. Owing to the abundant catechol groups, PEDOT can be assembled on the surface of ZIF-71 to create a redox-active system. The core-shell nanoparticles could act as a redox-active nanofiller to develop a conductive polyacrylamide (PAM) hydrogel with energy-storage properties. The core-shell PEDOT@PZIF-71 system provides a mussel-inspired environment in the hydrogel matrix and endows the hydrogel with stretchability and adhesiveness. The hydrogel can be applied as a functional electrode for both bioelectronics and supercapacitors. Moreover, this hydrogel exhibits favorable biocompatibility and can be implanted in vivo for biosignal measurement without causing inflammation. This redox-active core-shell PEDOT@PZIF-71 system provides a promising strategy for the design of hydrogel-based wearable electronic devices.

Oral polyphenol-armored nanomedicine for targeted modulation of gut microbiota-brain interactions in colitis.

Developing oral nanomedicines that suppress intestinal inflammation while modulating gut microbiota and brain interactions is essential for effectively treating inflammatory bowel disease. Here, we report an oral polyphenol-armored nanomedicine based on tumor necrosis factor-α (TNF-α)-small interfering RNA and gallic acid-mediated graphene quantum dot (GAGQD)-encapsulated bovine serum albumin nanoparticle, with a chitosan and tannin acid (CHI/TA) multilayer. Referred to "armor," the CHI/TA multilayer resists the harsh environment of the gastrointestinal tract and adheres to inflamed colon sites in a targeted manner. TA provides antioxidative stress and prebiotic activities that modulate the diverse gut microbiota. Moreover, GAGQD protected TNF-α-siRNA delivery. Unexpectedly, the armored nanomedicine suppressed hyperactive immune responses and modulated bacterial gut microbiota homeostasis in a mouse model of acute colitis. Notably, the armored nanomedicine alleviated anxiety- and depression-like behaviors and cognitive impairment in mice with colitis. This armor strategy sheds light on the effect of oral nanomedicines on bacterial gut microbiome-brain interactions.