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.

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.

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.

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.

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.

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.

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.

Whole genome sequencing identifies structural variants contributing to hematologic traits in the NHLBI TOPMed program.

Genome-wide association studies have identified thousands of single nucleotide variants and small indels that contribute to variation in hematologic traits. While structural variants are known to cause rare blood or hematopoietic disorders, the genome-wide contribution of structural variants to quantitative blood cell trait variation is unknown. Here we utilized whole genome sequencing data in ancestrally diverse participants of the NHLBI Trans Omics for Precision Medicine program (N = 50,675) to detect structural variants associated with hematologic traits. Using single variant tests, we assessed the association of common and rare structural variants with red cell-, white cell-, and platelet-related quantitative traits and observed 21 independent signals (12 common and 9 rare) reaching genome-wide significance. The majority of these associations (N = 18) replicated in independent datasets. In genome-editing experiments, we provide evidence that a deletion associated with lower monocyte counts leads to disruption of an S1PR3 monocyte enhancer and decreased S1PR3 expression.

Highly Efficient One-Step Tagging of Endogenous Genes in Primary Cells Using CRISPR-Cas Ribonucleoproteins.

Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein complexes and chemically modified donor DNA into cells. Upon CRISPR-Cas9 blunt-end double-strand breaks, highly efficient site-specific insertion of genetic materials (3 × FLAG or eGFP) was achieved in both cell lines and primary cells. We further optimized the gene-tagging efficiency and precision by using CRISPR-Cas12a, which produces a staggered cut with a 5' overhang and thus enables precise ligation of DNA donors with a complementary 3' overhang. With high efficiency and flexibility, this platform would be extremely useful for multiplex endogenous genes tagging and further exploration of protein functions in various cell types.

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.

Convergent lines of evidence supporting involvement of NFKB1 in schizophrenia.

OBJECTIVES: NFKB1 was associated with treatment-refractory schizophrenia (SZ) and response to antipsychotics; however, the underlying mechanisms through which NFKB1 confers its risk for SZ are largely unknown. We aimed to investigate the potential role of NFKB1 in SZ. METHODS: In the present study, we investigated the association of the risk SNP rs230529 of NFKB1 with gray matter density and with NFKB1 mRNA levels in various human brain regions. The spatiotemporal expression pattern of NFKB1 in human brains was explored. We constructed a miRNA-NFKB1-target gene regulatory network and analyzed its druggability through targeting NFKB1 for SZ treatment. RESULTS: NFKB1 showed the highest expression levels in the cerebellum, in which these levels were stratified by genotypes of rs230529. Interestingly, the allelic state of rs230529 was significantly associated with regional gray matter density in multiple brain regions (including the cerebellum), which also differed between patients with schizophrenia and controls. Furthermore, regulatory targets of NFKB1 were enriched among SZ susceptibility genes. A substantial proportion of NFKB1 target genes were subject to combinatorial regulation by NFKB1 and miRNAs, constituting a hybrid NFKB1-miRNA-gene regulatory network. Some components of this network showed expression changes relevant to both the disease and the treatment. Finally, we detected the dynamic changes of NFKB1-miR-155-5p-GSK3B and NFKB1-miR-155-5p/let-7a-5p-IL6 networks in course of the treatment of SZ. CONCLUSION: Taken together, our findings support the involvement of NFKB1-mediated dysregulation in the development of SZ.

Shared genetic liability between major depressive disorder and osteoarthritis.

AIMS: Deciphering the genetic relationships between major depressive disorder (MDD) and osteoarthritis (OA) may facilitate an understanding of their biological mechanisms, as well as inform more effective treatment regimens. We aim to investigate the mechanisms underlying relationships between MDD and OA in the context of common genetic variations. METHODS: Linkage disequilibrium score regression was used to test the genetic correlation between MDD and OA. Polygenic analysis was performed to estimate shared genetic variations between the two diseases. Two-sample bidirectional Mendelian randomization analysis was used to investigate causal relationships between MDD and OA. Genomic loci shared between MDD and OA were identified using cross-trait meta-analysis. Fine-mapping of transcriptome-wide associations was used to prioritize putatively causal genes for the two diseases. RESULTS: MDD has a significant genetic correlation with OA (rg = 0.29) and the two diseases share a considerable proportion of causal variants. Mendelian randomization analysis indicates that genetic liability to MDD has a causal effect on OA (bxy = 0.24) and genetic liability to OA conferred a causal effect on MDD (bxy = 0.20). Cross-trait meta-analyses identified 29 shared genomic loci between MDD and OA. Together with fine-mapping of transcriptome-wide association signals, our results suggest that Estrogen Receptor 1 (ESR1), SRY-Box Transcription Factor 5 (SOX5), and Glutathione Peroxidase 1 (GPX1) may have therapeutic implications for both MDD and OA. CONCLUSION: The study reveals substantial shared genetic liability between MDD and OA, which may confer risk for one another. Our findings provide a novel insight into phenotypic relationships between MDD and OA. Cite this article: Bone Joint Res 2022;11(1):12-22.

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.

Genetic evidence suggests posttraumatic stress disorder as a subtype of major depressive disorder.

BACKGROUNDMajor depressive disorder (MDD) and posttraumatic stress disorder (PTSD) are highly comorbid and exhibit strong correlations with one another. We aimed to investigate mechanisms of underlying relationships between PTSD and 3 kinds of depressive phenotypes, namely, MDD, depressed affect (DAF), and depression (DEP, including both MDD and the broad definition of depression).METHODSGenetic correlations between PTSD and the depressive phenotypes were tested using linkage disequilibrium score regression. Polygenic overlap analysis was used to estimate shared and trait-specific causal variants across a pair of traits. Causal relationships between PTSD and the depressive phenotypes were investigated using Mendelian randomization. Shared genomic loci between PTSD and MDD were identified using cross-trait meta-analysis.RESULTSGenetic correlations of PTSD with the depressive phenotypes were in the range of 0.71-0.80. The estimated numbers of causal variants were 14,565, 12,965, 10,565, and 4,986 for MDD, DEP, DAF, and PTSD, respectively. In each case, causal variants contributing to PTSD were completely or largely covered by causal variants defining each of the depressive phenotypes. Mendelian randomization analysis indicated that the genetically determined depressive phenotypes confer a causal effect on PTSD (b = 0.21-0.31). Notably, genetically determined PTSD confers a causal effect on DEP (b = 0.14) and DAF (b = 0.15), but not MDD. Cross-trait meta-analysis of MDD and PTSD identified 47 genomic loci, including 29 loci shared between PTSD and MDD.CONCLUSIONEvidence from shared genetics suggests that PTSD is a subtype of MDD. This study provides support to the efforts in reducing diagnostic heterogeneity in psychiatric nosology.FUNDINGThe National Key Research and Development Program of China and the National Natural Science Foundation of China.

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.

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.

Editing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation.

Genome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.