Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.

Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.

Systematical analysis reveals a strong cancer relevance of CREB1-regulated genes.

The transcription factor cyclic-AMP response element-binding protein 1 (CREB1) responds to cAMP level and controls the expression of target genes, which regulates nutrition partitioning. The promoters of CREB1-targeted genes responsive to cAMP have been extensively investigated and characterized with the presence of both cAMP response element and TATA box. Compelling evidence demonstrates that CREB1 also plays an essential role in promoting tumor development. However, only very few genes required for cell survival, proliferation and migration are known to be constitutively regulated by CREB1 in tumors. Their promoters mostly do not harbor any cAMP response element. Thus, it is very likely that CREB1 regulates the expressions of distinct sets of target genes in normal tissues and tumors. The whole gene network constitutively regulated by CREB1 in tumors has remained unrevealed. Here, we employ a systematical and integrative approach to decipher this gene network in the context of both tissue cultured cancer cells and patient samples. We combine transcriptomic, Rank-Rank Hypergeometric Overlap, and Chipseq analysis, to define and characterize CREB1-regulated genes in a multidimensional fashion. A strong cancer relevance of those top-ranked targets, which meet the most stringent criteria, is eventually verified by overall survival analysis of cancer patients. These findings strongly suggest the importance of genes constitutively regulated by CREB1 for their implicative involvement in promoting tumorigenesis.

Identification and functional characterization of mutations in LPL gene causing severe hypertriglyceridaemia and acute pancreatitis.

Hypertriglyceridaemia is a very rare disorder caused by the mutations of LPL gene, with an autosomal recessive mode of inheritance. Here, we identified two unrelated Chinese patients manifested with severe hypertriglyceridaemia and acute pancreatitis. The clinical symptoms of proband 1 are more severe than proband 2. Whole exome sequencing and Sanger sequencing were performed. Functional analysis of the identified mutations has been done. Whole exome sequencing identified two pairs of variants in LPL gene in the proband 1 (c.162C>A and c.1322+1G>A) and proband 2 (c.835C>G and c.1322+1G>A). The substitution (c.162C>A) leads to the formation of a truncated (p.Cys54*) LPL protein. The substitution (c.835C>G) leads to the replacement of leucine to valine (p.Leu279Val). The splice donor site mutation (c.1322+1G>A) leads to the formation of alternative transcripts with the loss of 134 bp in exon 8 of the LPL gene. The proband 1 and his younger son also harbouring a heterozygous variant (c.553G>T; p.Gly185Cys) in APOA5 gene. The relative expression level of the mutated LPL mRNA (c.162C>A, c.835C>G and c.1322+1G>A) showed significant differences compared to wild-type LPL mRNA, suggesting that all these three mutations affect the transcription of LPL mRNA. These three mutations (c.162C>A, c.835C>G and c.1322+1G>A) showed noticeably decreased LPL activity in cell culture medium but not in cell lysates. Here, we identified three mutations in LPL gene which causes severe hypertriglyceridaemia with acute pancreatitis in Chinese patients. We also described the significance of whole exome sequencing for identifying the candidate gene and disease-causing mutation in patients with severe hypertriglyceridaemia and acute pancreatitis.

Identification of the transcriptome signatures and immune-inflammatory responses in postmenopausal osteoporosis.

Postmenopausal osteoporosis is the most common type of osteoporosis in women. To date, little is known about their transcriptome signatures, although biomarkers from peripheral blood mononuclear cells are attractive for postmenopausal osteoporosis diagnoses. Here, we performed bulk RNA sequencing of 206 samples (124 postmenopausal osteoporosis and 82 normal samples) and described the clinical phenotypic characteristics of postmenopausal women. We then highlighted the gene set enrichment analyses between the extreme T-score group and the heathy control group, revealing that some immune-inflammatory responses were enhanced in postmenopausal osteoporosis, with representative pathways including the mitogen-activated protein kinase (NES = 1.6, FDR <0.11) pathway and B_CELL_RECEPTOR (NES = 1.69, FDR <0.15) pathway. Finally, we developed a combined risk prediction model based on lasso-logistic regression to predict postmenopausal osteoporosis, which combined eleven genes (PTGS2, CXCL16, NECAP1, RPS23, SSR3, CD74, IL4R, BTBD2, PIGS, LILRA2, MAP3K11) and three pieces of clinical information (age, procollagen I N-terminal propeptide, ß isomer of C-terminal telopeptide of type I) and provided the best prediction ability (AUC = 0.97). Taken together, this study filled a gap in the large-scale transcriptome signature profiles and revealed the close relationship between immune-inflammatory responses and postmenopausal osteoporosis, providing a unique perspective for understanding the occurrence and development of postmenopausal osteoporosis.

A porcine brain-wide RNA editing landscape.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is an essential post-transcriptional modification. Although hundreds of thousands of RNA editing sites have been reported in mammals, brain-wide analysis of the RNA editing in the mammalian brain remains rare. Here, a genome-wide RNA-editing investigation is performed in 119 samples, representing 30 anatomically defined subregions in the pig brain. We identify a total of 682,037 A-to-I RNA editing sites of which 97% are not identified before. Within the pig brain, cerebellum and olfactory bulb are regions with most edited transcripts. The editing level of sites residing in protein-coding regions are similar across brain regions, whereas region-distinct editing is observed in repetitive sequences. Highly edited conserved recoding events in pig and human brain are found in neurotransmitter receptors, demonstrating the evolutionary importance of RNA editing in neurotransmission functions. Although potential data biases caused by age, sex or health status are not considered, this study provides a rich resource to better understand the evolutionary importance of post-transcriptional RNA editing.

Efficient correction of Duchenne muscular dystrophy mutations by SpCas9 and dual gRNAs.

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning.

The design of CRISPR gRNAs requires accurate on-target efficiency predictions, which demand high-quality gRNA activity data and efficient modeling. To advance, we here report on the generation of on-target gRNA activity data for 10,592 SpCas9 gRNAs. Integrating these with complementary published data, we train a deep learning model, CRISPRon, on 23,902 gRNAs. Compared to existing tools, CRISPRon exhibits significantly higher prediction performances on four test datasets not overlapping with training data used for the development of these tools. Furthermore, we present an interactive gRNA design webserver based on the CRISPRon standalone software, both available via https://rth.dk/resources/crispr/ . CRISPRon advances CRISPR applications by providing more accurate gRNA efficiency predictions than the existing tools.

An atlas of the protein-coding genes in the human, pig, and mouse brain.

The brain, with its diverse physiology and intricate cellular organization, is the most complex organ of the mammalian body. To expand our basic understanding of the neurobiology of the brain and its diseases, we performed a comprehensive molecular dissection of 10 major brain regions and multiple subregions using a variety of transcriptomics methods and antibody-based mapping. This analysis was carried out in the human, pig, and mouse brain to allow the identification of regional expression profiles, as well as to study similarities and differences in expression levels between the three species. The resulting data have been made available in an open-access Brain Atlas resource, part of the Human Protein Atlas, to allow exploration and comparison of the expression of individual protein-coding genes in various parts of the mammalian brain.