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Nature Biotechnology asks a selection of leaders from across biotech to look at the future of the sector and make some predictions for the coming years.

African Swine Fever (ASF) is a viral disease with exceptionally high lethality in domestic pigs and wild boar worldwide1, 2, without any effective vaccine or drug to prevent its spread. In this study, we established a multiplexable CRISPR-Cas-gRNA system targeting 13 genomic loci in the ASF virus genome that could prevent viral replication by cutting its genome. Furthermore, we generated pig strains expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing and demonstrated that the gene-edited pigs were more resistant to ASFV infection and less likely to spread the virus upon infection. As far as we know, our study presents the first living organism generated via germline editing to demonstrate resistance to viral infection via CRISPR-Cas. We anticipate our work to be helpful for both agricultural and biomedical applications, such as xenotransplantation.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Accurate chromosome segregation relies on correct chromosome-microtubule interactions within a stable bipolar spindle apparatus. Thus, exposure to spindle disrupting compounds can impair meiotic division and genomic stability in oocytes. The endocrine disrupting activity of bisphenols such as bisphenol A (BPA) is well recognized, yet their damaging effects on spindle microtubules (MTs) is poorly understood. Here, we tested the effect(s) of acute exposure to BPA and bisphenol F (BPF) on assembled spindle stability in ovulated oocytes. Brief (4 h) exposure to increasing concentrations (5, 25, and 50 µg/mL) of BPA or BPF disrupted spindle organization in a dose-dependent manner, resulting in significantly shorter spindles with highly unfocused poles and fragmented pericentrin. The chromosomes remained congressed in an abnormally elongated metaphase-like configuration, yet normal end-on chromosome-MT attachments were reduced in BPF-treated oocytes. Live-cell imaging revealed a rapid onset of bisphenol-mediated spindle MT disruption that was reversed upon compound removal. Moreover, MT stability and regrowth were impaired in BPA-exposed oocytes, with few cold-stable MTs and formation of multipolar spindles upon MT regrowth. MT-associated kinesin-14 motor protein (HSET/KIFC1) labeling along the spindle was also lower in BPA-treated oocytes. Conversely, cold stable MTs and HSET labeling persisted after BPF exposure. Notably, inhibition of Aurora Kinase A limited bisphenol-mediated spindle pole widening, revealing a potential interaction. These results demonstrate rapid MT disrupting activity by bisphenols, which is highly detrimental to meiotic spindle stability and organization. Moreover, we identify an important link between these defects and altered distribution of key spindle associated factors as well as Aurora Kinase A activity.

Water hyacinth is a major aquatic plant in ecological restoration which propagates rapidly, whereas its biomass waste lacks value-added utilization routes. To address this problem, we put forth an innovative two-step carbonization strategy to convert water hyacinth to catalyst for isomerization of glucose to fructose. Through combining the hydrothermal carbonization and pyrolysis, catalyst morphology including its carbon substrate and calcium salts was successfully engineered. The prepared hydrochar-based catalyst presented an outstanding catalytic performance, the optimal of which could obtain 31% fructose yield with 89% selectivity at 120 °C for 45 min in water and maintain the reactivity for at least three runs. The catalytic reactivity was derived from the crystallization of endogenous alkaline earth calcium in water hyacinth, which was comparable to catalysts doped with expensive metals. Besides, the equipment and energy requirements for preparation were quite low-demanding (calcined only at 400 °C for 1 h). This study not only pioneers a sustainable way to upcycle aquatic biomass, but also invents a low-cost and efficient catalyst for biorefinery through the production of engineered carbon.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Germline editing, the process by which the genome of an individual is edited in such a way that the change is heritable, has been applied to a wide variety of animals [D. A. Sorrell, A. F. Kolb, Biotechnol. Adv. 23, 431–469 (2005); D. Baltimore et al., Science 348, 36–38 (2015)]. Because of its relevancy in agricultural and biomedical research, the pig genome has been extensively modified using a multitude of technologies [K. Lee, K. Farrell, K. Uh, Reprod. Fertil. Dev. 32, 40–49 (2019); C. Proudfoot, S. Lillico, C. Tait-Burkard, Anim. Front. 9, 6–12 (2019)]. In this perspective, we will focus on using pigs as the model system to review the current methodologies, applications, and challenges of mammalian germline genome editing. We will also discuss the broad implications of animal germline editing and its clinical potential.

CAR-T cells expressing chimeric antigen receptors (CAR) have shown clinical success for treating hematologic malignancies, but their efficacy in solid tumors has been limited, largely due to tumor heterogeneity and immunosuppressive tumor microenvironment (TME). Natural Killer (NK) cells derived from human induced pluripotent stem cells (hiPSC) hold great potential to become the next-generation cell therapy products because of their established safety profile and their unique biological activities, especially their potential effectiveness for targeting solid tumor. In contrast to T cells, NK cells exert anti-tumor activity without requiring antigen recognition through MHC complex; they are instead activated by calculated signaling through ligation of multiple activating and inhibitory receptors. As various solid tumors highly express activation ligands for NK cells, we reason that enhanced expression of activation receptors on NK cells could improve their killing activity towards solid tumors. Here, we report that “super NK cells” with more than 10 folds higher expression of NK cell receptors such as NKG2D and NCRs derived from engineered hiPSC show improved cytotoxicity against solid tumors compared with non-engineered ones. The “super NK cells” can achieve more than ten rounds of serial killing against both K562 and THP-1 cell lines in vitro. More importantly, the “super NK cells” show improved cytotoxicity to multiple solid tumors in both in vitro system and in vivo xenograft mouse models. In summary, we have engineered hiPSCs which can be differentiated into "super NK cells" as a potential cell therapy product for improved efficacy against solid tumors. Citation Format: Yiyun Wang, Xiangjun He, Linqin Wang, Mi Shao, Yanan Yue, Yangbin Gao, George Church, He Huang, Luhan Yang. "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3155.

Endocrine dyscrasia generally is the priority issue bothering the modern woman. To research the regulating effect of the long-term high-intensity aerobic exercise on the secretion of endocrine horm...

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO·3KO·9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles.In vitroassays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO·3KO·9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism.One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

Background: Numerous case-control studies have demonstrated that single nucleotide polymorphism (SNP) loci in the TLR4 3'-untranslated region (UTR) are associated with multiple inflammatory disorders in various populations, particularly in Asians. However, the distribution frequency of polymorphisms in different Asian races and its effects on TLR4 expression remain unclear. Here, we extracted variants from the 1000 Genomes project database and analyzed the SNPs and haplotypes genetic diversity of TLR4 3'-UTR in 10 Asian populations, and then assessed the biological effects of the altofrequent polymorphic loci on TLR4 expression by luciferase assay. Results: Total 6 SNPs were identified as true polymorphic loci with minor allele frequencies (MAFs) ≥ 1% in Asian populations, including rs41426344, rs7869402, rs11536889, rs7873784, rs11536891, and rs11536896. Considering 4 Tag SNPs, we inferred 5 haplotypes that present a Asiatic frequency higher than 1%. It is noteworthy that rs41426344 is unique to East Asian populations, and the H-5 haplotype frequency was reduced when analyzing pooled data from East and South Asian populations. The MAFs of rs7869402 and rs11536889, and H-2 and H-4 frequencies significantly differed between the populations (P < 0.001). We constructed a pGL3-3494-3UTR luciferase plasmid to simulate TLR4 gene structure in vivo, and used PCR-mediated site-directed mutagenesis to construct a series of mutant luciferase constructs corresponding to the six SNPs. In addition, we identified that TLR4 mRNA was selectively expressed in SiHa and THP-1 cell lines, but not in C33A, Hela, and 293T cell lines. The luciferase activity of constructs containing rs7869402 T allele and rs11536889 C allele increased significantly upon LPS or IL-6 stimulation in THP-1 and SiHa cells. Conclusions: The distributions of SNPs and haplotypes in TLR4 3'-UTR were significantly different among Asian populations. The biological effects of rs7869402 and rs11536889 on TLR4 are significant clues that revealing its critical role in harmful TLR4-mediated responses. These results provided a reminder for future investigation on TLR4 related inflammatory diseases susceptibility.

Since efficient isomerization of glucose to fructose is vital for valorizing cellulose fraction of biomass to value-added chemicals, an approach of engineering aluminum-hydrochar catalyst by impregnating aluminum on swollen cellulose derived hydrochar has been studied. The results showed that Al-hydrochar calcinated at 300°C achieved fructose yield of 26.3% in acetone/H2O reaction medium. It was found that the amorphous Al structures with nano-size on the surface of the carbon microspheres were the major contributor of the catalytic activity on glucose to fructose isomerization, while the formation of Al crystal had an inhibition effect on glucose isomerization. The deactivation study of Al-hydrochar catalysts showed the exfoliation of colloidal carbon containing aluminum active catalytic sites. This finding provides a novel strategy for efficient isomerization of glucose by Al-hydrochar prepared through hydrothermal carbonization and mild calcination activation process.

Ai - database integration is an effective application for many artificial intelligence technologies as well as major technologies for database development, and for the next generation of computing It supports intelligent information systems based on distributed cooperative tasks. The relationship between AI and large data and the potential values of Bayes in AI are described.

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

For designing the heat exchanger to supply necessary anti-freeze heating in the inlet wellhead of a mine, the specific heat demand there and the state of the art of the heat pipe technologies for waste heat recovery are reviewed. According to the existing conditions in a mine located in a cold region, a heat pipe heat exchanger was designed, the feasibility of this heat pipe heat exchanger was theoretically investigated, and the influence of the heat exchange efficiency on the effect of the heat exchanger was analyzed. Then the necessary guarantee measures and the implementation scheme for sufficient heat supply under the extremely cold weather conditions were pointed out. Finally, the feasibility of this heat pipe heat exchange technology is verified by the actual preliminary operation performance and the key technical problems of its application are clarified.

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.

Beta cell replacement has the potential to restore euglycemia in patients with insulin dependent diabetes. While great progress has been made in establishing allogeneic islet transplantation from deceased donors as the standard of care for those with the most labile diabetes, it is also clear that the deceased donor organ supply cannot possibly treat all those who could benefit from restoration of a normal beta cell mass, especially if immunosuppression were not required. Against this background, the International Pancreas and Islet Transplant Association (IPITA) in collaboration with the Harvard Stem Cell Institute (HSCI), the Juvenile Diabetes Research Foundation (JDRF), and the Helmsley Foundation held a 2-day Key Opinion Leaders Meeting in Boston in 2016 to bring together experts in generating and transplanting beta cells derived from stem cells. The following summary highlights current technology, recent significant breakthroughs, unmet needs and roadblocks to stem-cell-derived beta cell therapies, with the aim of spurring future preclinical collaborative investigations and progress toward the clinical application of stem cell-derived beta cells.

Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders and diseases such as cancer. However, there is a lack of genetic engineering tool to recapitulate these features. Here, we report the discovery that delivery of pairs of CRISPR/Cas9 guide RNAs into human cells generate functional eccDNAs and ring chromosomes. We generated a dual-fluorescence eccDNA biosensor system, which allows us to study CRISPR deletion, inversion, and circularization of genes inside cells. Analysis after CRISPR editing at intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts reveal that CRISPR deleted DNA readily form eccDNA in human cells. DNA in sizes from a few hundred base pairs up to a 47.4 megabase-sized ring chromosome (chr18) can be circularized. Our discoveries advance and expand CRISPR-Cas9 technology applications for genetic engineering, modeling of human diseases, and chromosome engineering.One Sentence Summary: CRISPR circularization of DNA offers new tools for studying eccDNA biogenesis, function, chromosome engineering, and synthetic biology.

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.