Massively targeted evaluation of therapeutic CRISPR off-targets in cells.

Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

A Brief Overview of Global Trends in MSC-Based Cell Therapy.

Human mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells or medicinal signaling cells, are important adult stem cells for regenerative medicine, largely due to their regenerative characteristics such as self-renewal, secretion of trophic factors, and the capability of inducing mesenchymal cell lineages. MSCs also possess homing and trophic properties modulating immune system, influencing microenvironment around damaged tissues and enhancing tissue repair, thus offering a broad perspective in cell-based therapies. Therefore, it is not surprising that MSCs have been the broadly used adult stem cells in clinical trials. To gain better insights into the current applications of MSCs in clinical applications, we perform a comprehensive review of reported data of MSCs clinical trials conducted globally. We summarize the biological effects and mechanisms of action of MSCs, elucidating recent clinical trials phases and findings, highlighting therapeutic effects of MSCs in several representative diseases, including neurological, musculoskeletal diseases and most recent Coronavirus infectious disease. Finally, we also highlight the challenges faced by many clinical trials and propose potential solutions to streamline the use of MSCs in routine clinical applications and regenerative medicine.

A Bacterial Genome and Culture Collection of Gut Microbial in Weanling Piglet.

The microbiota hosted in the pig gastrointestinal tract are important to health of this biomedical model. However, the individual species and functional repertoires that make up the pig gut microbiome remain largely undefined. Here we comprehensively investigated the genomes and functions of the piglet gut microbiome using culture-based and metagenomics approaches. A collection included 266 cultured genomes and 482 metagenome-assembled genomes (MAGs) that were clustered to 428 species across 10 phyla was established. Among these clustered species, 333 genomes represent potential new species. Less matches between cultured genomes and MAGs revealed a substantial bias for the acquisition of reference genomes by the two strategies. Glycoside hydrolases was the dominant category of carbohydrate-active enzymes. Four-hundred forty-five secondary metabolite biosynthetic genes were predicted from 292 genomes with bacteriocin being the most. Pan genome analysis of Limosilactobacillus reuteri uncover the biosynthesis of reuterin was strain-specific and the production was experimentally determined. This study provides a comprehensive view of the microbiome composition and the function landscape of the gut of weanling piglets and a valuable bacterial resource for further experimentations. IMPORTANCE The microorganism communities resided in mammalian gastrointestinal tract impacted the health and disease of the host. Our study complements metagenomic analysis with culture-based approach to establish a bacteria and genome collection and comprehensively investigate the microbiome composition and function of the gut of weanling piglets. We provide a valuable resource for further study of gut microbiota of weanling piglet and development of probiotics for prevention of disease.

Comparative analysis of clonal evolution among patients with right- and left-sided colon and rectal cancer.

Tumor multiregion sequencing reveals intratumor heterogeneity (ITH) and clonal evolution playing a key role in tumor progression and metastases. Large-scale high-depth multiregional sequencing of colorectal cancer, comparative analysis among patients with right-sided colon cancer (RCC), left-sided colon cancer (LCC), and rectal cancer (RC), as well as the study of lymph node metastasis (LN) with extranodal tumor deposits (ENTDs) from evolutionary perspective remain weakly explored. Here, we recruited 68 patients with RCC (18), LCC (20), and RC (30). We performed high-depth whole-exome sequencing of 206 tumor regions including 176 primary tumors, 19 LN, and 11 ENTD samples. Our results showed ITH with a Darwinian pattern of evolution and the evolution pattern of LCC and RC was more complex and divergent than RCC. Genetic and evolutionary evidences found that both LN and ENTD originated from different clones. Moreover, ENTD was a distinct entity from LN and evolved later.

A Chromosome-Level Genome Assembly of the Anglerfish Lophius litulon.

Anglerfishes are a highly diverse group of species with unique characteristics. Here, we report the first chromosome-level genome of a species in the order Lophiiformes, the yellow goosefish (Lophius litulon), obtained by whole genome shotgun sequencing and high-throughput chromatin conformation capture. Approximately 97.20% of the assembly spanning 709.23 Mb could be anchored to 23 chromosomes with a contig N50 of 164.91 kb. The BUSCO value was 95.4%, suggesting that the quality of the assembly was high. A comparative gene family analysis identified expanded and contracted gene families, and these may be associated with adaptation to the benthic environment and the lack of scales in the species. A majority of positively selected genes were related to metabolic processes, suggesting that digestive and metabolic system evolution expanded the diversity of yellow goosefish prey. Our study provides a valuable genetic resource for understanding the mechanisms underlying the unique features of the yellow goosefish and for investigating anglerfish evolution.

Family-Based Whole Genome Sequencing Identified Novel Variants in ABCA5 Gene in a Patient with Idiopathic Ventricular Tachycardia.

Idiopathic ventricular tachycardia (IVT) is the major cause of sudden cardiac death. Patients with IVT were usually manifested without structural heart disease. In this present study, we performed family-based whole genome sequencing (WGS) and Sanger sequencing for a 5-year-old Chinese boy with IVT and all the unaffected family members in order to identify the candidate gene and disease-causing mutation underlying the disease phenotype. Results showed that a novel heterozygous single-nucleotide duplication (c.128dup) and a novel heterozygous missense (c.3328A > G) variant in ABCA5 gene were identified in the proband. The single-nucleotide duplication (c.128dupT), inherited from his father and patrilineal grandfather, leads to a frameshift which results into the formation of a truncated ABCA5 protein of 50 (p.Leu43Phefs*8) amino acids. Hence, it is a loss-of-function mutation. The missense (c.3328A > G) variant, inherited from his mother, leads to the replacement of isoleucine by valine at the position of 1110 (p.Ile1110Val) of the ABCA5 protein. Multiple sequence alignment showed that p.Ile1110 is evolutionarily conserved among several species indicating both the structural and functional significance of the p.Ile1110 residue in the wild-type ABCA5 protein. Quantitative RT-PCR showed that the ABCA5 mRNA expression levels were decreased in the proband. These two novel variants of ABCA5 gene were co-segregated well among all the members of this family. Our present study also strongly supports the importance of using family-based whole genome sequencing for identifying novel candidate genes associated with IVT.

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.

Whole exome sequencing identified a homozygous novel variant in CEP290 gene causes Meckel syndrome.

Meckel syndrome (MKS) is a pre- or perinatal multisystemic ciliopathic lethal disorder with an autosomal recessive mode of inheritance. Meckel syndrome is usually manifested with meningo-occipital encephalocele, polycystic kidney dysplasia, postaxial polydactyly and hepatobiliary ductal plate malformation. Germline variants in CEP290 cause MKS4. In this study, we investigated a 35-years-old Chinese female who was 17+1 weeks pregnant. She had a history of adverse pregnancy of having foetus with multiple malformations. We performed ultrasonography and identified the foetus with occipital meningoencephalocele and enlarged cystic dysplastic kidneys. So, she decided to terminate her pregnancy and further genetic molecular analysis was performed. We identified the aborted foetus without postaxial polydactyly. Histological examination of foetal kidney showed cysts in kidney and thinning of the renal cortex with glomerular atrophy. Whole exome sequencing identified a novel homozygous variant (c.2144T>G; p.L715* ) in exon 21 of the CEP290 in the foetus. Sanger sequencing confirmed that both the parents of the foetus were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters of the foetus as well as in the 100 healthy individuals. Western blot analysis showed that this variant leads to the formation of truncated CEP290 protein with the molecular weight of 84 KD compared with the wild-type CEP290 protein of 290 KD. Hence, it is a loss-of-function variant. We also found that the mutant cilium appears longer in length than the wild-type cilium. Our present study reported the first variant of CEP290 associated with MKS4 in Chinese population.

Egg quality and egg albumen property of domestic chicken, duck, goose, turkey, quail, and pigeon.

Poultry eggs from different species varied significantly, due to their divergent process of evolution. However, the information on egg characteristics has been limited mostly to chicken. The current study compared the egg quality, albumen physical and nutritional property for domesticated chicken, duck, goose, turkey, quail, and pigeon. Egg quality traits among different species differed significantly, such as egg weight (from 11 to 139 g), egg shape (from 1.28 to 1.44), proportion of yolk (from 19.3 to 37.9%), and breaking strength (from 0.91 to 8.04 kg/cm2). For the physical property of albumen gel, pigeon egg was the most special one. The albumen gel of pigeon egg had a transparent appearance (A = 1.23), and it had the highest hardness (121.7 g) and water-holding capacity (96%) but a medium level of total cutting work (440 g⋅s). Hence, it was easy for deformation but was hard to cut off when external force was applied. For nutritional compositions of egg albumen, goose egg had the highest moisture (89.21%) and lowest crude protein (8.5%) contents. Specific to amino acid, glutamic acid was found the most abundant in albumen of all six species. Egg albumen of turkey had the highest quantity of essential amino acids (EAA) and total amino acids (TAA), while duck and goose had relatively higher EAA/TAA ratios. Both PCA and cluster analysis revealed the high similarity of amino acid composition between duck and goose, and among quail, turkey, and chicken. The comparative data will improve the understanding of egg qualities of major poultry species and can be helpful in technological application of egg albumen.

The first chromosome-level genome for a marine mammal as a resource to study ecology and evolution.

Marine mammals are important models for studying convergent evolution and aquatic adaption, and thus reference genomes of marine mammals can provide evolutionary insights. Here, we present the first chromosome-level marine mammal genome assembly based on the data generated by the BGISEQ-500 platform, for a stranded female sperm whale (Physeter macrocephalus). Using this reference genome, we performed chromosome evolution analysis of the sperm whale, including constructing ancestral chromosomes, identifying chromosome rearrangement events and comparing with cattle chromosomes, which provides a resource for exploring marine mammal adaptation and speciation. We detected a high proportion of long interspersed nuclear elements and expanded gene families, and contraction of major histocompatibility complex region genes which were specific to sperm whale. Using comparisons with sheep and cattle, we analysed positively selected genes to identify gene pathways that may be related to adaptation to the marine environment. Further, we identified possible convergent evolution in aquatic mammals by testing for positively selected genes across three orders of marine mammals. In addition, we used publicly available resequencing data to confirm a rapid decline in global population size in the Pliocene to Pleistocene transition. This study sheds light on the chromosome evolution and genetic mechanisms underpinning sperm whale adaptations, providing valuable resources for future comparative genomics.

Divergent Proteome Patterns of Egg Albumen from Domestic Chicken, Duck, Goose, Turkey, Quail and Pigeon.

To uncover a diversity of genetic and biological unknowns, a comprehensive and comparative proteomic analysis is performed on egg albumen of domestic chicken, duck, goose, turkey, quail, and pigeon with tandem mass tags quantification technology. In this study, a total of 148, 138, 150, 162, 183, and 179 proteins are identified in egg albumen of the above six species, respectively. Venn plots, PCA, and cluster analysis all reveal the highest similarity of protein composition between duck and goose (≈75%). Additionally, the six species have 52 proteins detected in common in the egg albumen. As revealed by GO and pathway analyses, the plausible functions of these highly conserved proteins are to provide a secure environment and prevent the early death of embryonic cells. Species-specific proteins such as haptoglobin in pigeon, serpin-like protein HMSD in duck, and ovodefensin in chicken are also screened and are likely associated with species-dependent biological processes. Furthermore, Enzyme Code analysis indicated egg albumen have abundant enzyme activity, with hydrolases accounting for more than half of the total enzymes. This study is the first to provide the proteome profiles of egg albumen for the major poultry species, which will be instructive for the understanding of species-specific biological problems with egg albumen.