The genetics of human infertility by functional interrogation of SNPs in mice

Infertility is a prevalent health issue, affecting ∼15% of couples of childbearing age. Nearly one-half of idiopathic infertility cases are thought to have a genetic basis, but the underlying causes are largely unknown. Traditional methods for studying inheritance, such as genome-wide association studies and linkage analyses, have been confounded by the genetic and phenotypic complexity of reproductive processes. Here we describe an association- and linkage-free approach to identify segregating infertility alleles, in which CRISPR/Cas9 genome editing is used to model putatively deleterious nonsynonymous SNPs (nsSNPs) in the mouse orthologs of fertility genes. Mice bearing “humanized” alleles of four essential meiosis genes, each predicted to be deleterious by most of the commonly used algorithms for analyzing functional SNP consequences, were examined for fertility and reproductive defects. Only a Cdk2 allele mimicking SNP rs3087335, which alters an inhibitory WEE1 protein kinase phosphorylation site, caused infertility and revealed a novel function in regulating spermatogonial stem cell maintenance. Our data indicate that segregating infertility alleles exist in human populations. Furthermore, whereas computational prediction of SNP effects is useful for identifying candidate causal mutations for diverse diseases, this study underscores the need for in vivo functional evaluation of physiological consequences. This approach can revolutionize personalized reproductive genetics by establishing a permanent reference of benign vs. infertile alleles.Despite the high incidence of infertility, autosomal genetic causes of infertility attributable to gametogenesis defects are poorly characterized. In males, the most common known genetic causes of infertility are Y chromosome microdeletions, thought to be responsible for 6–18% of nonobstructive azoospermia (NOA) or severe oligozoospermia cases (1). In females, most of the known genetic causes are linked to syndromes that also affect the soma (e.g., Kallmann, Turner) or the neuroendocrine axis (2). In rare cases, families segregating infertility alleles have been mapped by linkage (3–6). Several candidate gene resequencing studies have implicated mutations or SNPs as being causative for azoospermia (7–13), but in the absence of genetic data, only a few reports (e.g., ref. 14) have made a compelling case. Recently, hemizygous deletions of the TEX11 gene, presumably catalyzed by unequal recombination between repetitive elements in the locus, have been linked to maturation arrest and infertility in azoospermic men (15).Gene knockout and molecular genetic studies in mice have shown that germ cell development is genetically complex. A screen of the Mouse Genome Informatics database using MouseMine (www.mousemine.org) identifies 728 genes currently associated with infertility. Clearly, many more genes required for fertility remain to be identified. Furthermore, infertility is genetically heterogeneous; scores of distinct genes cause grossly identical phenotypes when mutated in mice (2, 16). This likely explains why genome-wide association studies (GWAS) have not been effective even in stratified cohorts, with only two reporting significant associations with NOA in Chinese populations (11, 17). Even if associations could be readily obtained, identification and validation of causative variants would remain problematic. Finally, the proportion of Mendelian infertilities that are caused by de novo mutations vs. segregating polymorphisms is unknown. Clearly, different approaches are needed to address the genetics of human infertility.Here we describe a reverse genetics approach for identifying infertility alleles segregating in human populations that does not require linkage or association data; rather, it combines in silico prediction of deleterious allelic variants with functional validation in CRISPR/Cas9-edited “humanized” mouse models. We modeled four nonsynonymous human SNPs (nsSNPs) in genes that are essential for meiosis in mice. Each of these nsSNPs has been predicted to be deleterious to protein function by several widely used algorithms. Only one of the nsSNPs was found to cause infertility, highlighting the importance of experimentally evaluating computationally predicted disease SNPs.     

CRISPR-Associated (CAS) Effectors Delivery via Microfluidic Cell-Deformation Chip

Identifying new and even more precise technologies for modifying and manipulating selectively specific genes has provided a powerful tool for characterizing gene functions in basic research and potential therapeutics for genome regulation. The rapid development of nuclease-based techniques such as CRISPR/Cas systems has revolutionized new genome engineering and medicine possibilities. Additionally, the appropriate delivery procedures regarding CRISPR/Cas systems are critical, and a large number of previous reviews have focused on the CRISPR/Cas9–12 and 13 delivery methods. Still, despite all efforts, the in vivo delivery of the CAS gene systems remains challenging. The transfection of CRISPR components can often be inefficient when applying conventional delivery tools including viral elements and chemical vectors because of the restricted packaging size and incompetency of some cell types. Therefore, physical methods such as microfluidic systems are more applicable for in vitro delivery. This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations.     

CRISPR/Cas9基因编辑技术在病原微生物中的应用

规律成簇间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)广泛存在于古细菌及细菌中是细菌在长期进化过程中形成的一种获得性免疫系统。近年来以该系统为基础,经过人工改造形成的一种新型基因编辑技术—CRISPR/Cas9在基因工程领域的应用越发广泛;该技术与前两代编辑技术相比,具有结构简单、成本低廉、实用价值较高等优点。自2012年在基因研究领域成功应用后,已成为当前关注度较高的基因编辑工具;该技术在多种真核生物的基因修饰中已得到成功应用,但在病原微生物上却报道较少。本文将从CRISPR/Cas9系统的结构、作用机制及其在病原微生物基因功能研究中的应用等几个方面进行综述为其深入研究奠定基础。     

On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles

Lipid nanoparticles (LNPs) are a clinically mature technology for the delivery of genetic medicines but have limited therapeutic applications due to liver accumulation. Recently, our laboratory developed selective organ targeting (SORT) nanoparticles that expand the therapeutic applications of genetic medicines by enabling delivery of messenger RNA (mRNA) and gene editing systems to non-liver tissues. SORT nanoparticles include a supplemental SORT molecule whose chemical structure determines the LNP’s tissue-specific activity. To understand how SORT nanoparticles surpass the delivery barrier of liver hepatocyte accumulation, we studied the mechanistic factors which define their organ-targeting properties. We discovered that the chemical nature of the added SORT molecule controlled biodistribution, global/apparent pK_a, and serum protein interactions of SORT nanoparticles. Additionally, we provide evidence for an endogenous targeting mechanism whereby organ targeting occurs via 1) desorption of poly(ethylene glycol) lipids from the LNP surface, 2) binding of distinct proteins to the nanoparticle surface because of recognition of exposed SORT molecules, and 3) subsequent interactions between surface-bound proteins and cognate receptors highly expressed in specific tissues. These findings establish a crucial link between the molecular composition of SORT nanoparticles and their unique and precise organ-targeting properties and suggest that the recruitment of specific proteins to a nanoparticle’s surface can enable drug delivery beyond the liver.

Nucleic acids that enable gene silencing (1, 2), expression (3–5), and editing (6–8) possess great potential for use as genetic medicines in multiple clinical settings including cancer (9, 10), inherited genetic disorders (11, 12), and infectious diseases (13–15). Due to the unfavorable pharmacokinetic properties of nucleic acids, viral and nonviral delivery approaches are used to facilitate nucleic acid delivery to target cells (16). Lipid nanoparticles (LNPs) represent the most clinically mature nonviral platform for the safe and efficacious delivery of genetic medicines. Indeed, LNPs were an enabling technology for the US Food and Drug Administration approval of the first small interfering RNA (siRNA) drug, Onpattro, in 2018 (17) and the messenger RNA (mRNA) vaccines currently being distributed for immunization against the SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic (18, 19). Despite this progress, intravenously (IV) administered LNPs typically accumulate in the liver and are internalized by liver hepatocytes, thereby greatly limiting the scope of their therapeutic applications (20).Recently, our laboratory overcame this challenge through the discovery of selective organ targeting (SORT), a strategy to rationally design nanoparticles for the extrahepatic delivery of mRNA and gene editing systems following IV administration (Fig. 1A and SI Appendix, Table S1) (21–24). Conventional LNPs are composed of four components: ionizable cationic lipids, amphipathic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids (25, 26). We found that augmenting conventional four-component LNPs for mRNA delivery to the liver with a fifth component (termed a SORT molecule) can alter the LNPs’ in vivo organ-targeting properties and lead to the extrahepatic delivery of mRNA. The SORT strategy was generalizable to multiple classes of LNPs and SORT molecules. Lung-, liver-, and spleen-targeting SORT LNPs could deliver diverse cargoes (nucleic acids and proteins) to achieve gene expression and CRISPR/Cas-based gene editing in therapeutically relevant cell types, including epithelial cells, endothelial cells, B cells, T cells, and hepatocytes (21–24). However, the mechanism of action remains undefined. Understanding the mechanism which enables SORT is important for optimizing delivery to currently targetable organs as well as extending the SORT concept to other tissue and cell types. Moreover, mechanistic understanding would establish a biological rationale for overcoming the delivery barrier of liver accumulation that currently hampers IV administered nanoparticles (27).Open in a separate windowFig. 1.SORT nanoparticles for tissue-specific mRNA delivery have unique biodistribution and ionization behavior. (A) By adding a fifth, supplemental SORT molecule to a conventional, four-component LNP (mDLNP: 23.8 mol % 5A2-SC8, 23.8 mol % DOPE, 47.6 mol % cholesterol, and 4.8 mol % C14-PEG2K), the tissue-specific activity of delivered mRNA changes based on the chemical structure of the included SORT molecule. An ionizable cationic lipid (DODAP) enhances liver-specific mRNA translation (liver SORT: 19 mol % 5A2-SC8, 19 mol % DOPE, 38 mol % cholesterol, 4 mol % C14-PEG2K, and 20 mol % DODAP), an anionic lipid (18PA) results in spleen-specific mRNA translation (spleen SORT: 16.7 mol % 5A2-SC8, 16.7 mol % DOPE, 33.3 mol % cholesterol, 3.3 mol % C14-PEG2K, and 30 mol % 18PA), and a cationic quaternary ammonium lipid (DOTAP) results in lung-specific mRNA translation (lung SORT: 11.9 mol % 5A2-SC8, 11.9 mol % DOPE, 23.8 mol % cholesterol, 2.4 mol % C14-PEG2K, and 50 mol % DOTAP). (B) Ex vivo fluorescence of Cy5-labeled mRNA in major organs extracted from C57BL/6 mice IV injected with SORT LNPs that incorporate increasing percentages of different SORT molecules (0.5 mg/kg mRNA/body weight, 6 h). (C) Relative average Cy5 fluorescence measured in the liver, lung, and spleen as a function of SORT molecule percent inclusion (0.5 mg/kg mRNA/body weight, n = 2). SORT molecules promote mRNA biodistribution to target organs. Data are shown as mean ± SEM. (D) Representative TNS assay curves for determining the apparent pK_a of SORT LNPs incorporating increasing percentages of ionizable cationic, anionic, or permanently cationic lipid SORT molecules. Apparent pK_a was defined as the point at which 50% of TNS fluorescence was achieved. (E) LNPs were assigned a tissue specificity index based on the tissues in which functional luciferase mRNA was detected. Intermediate tissue specificity indexes represent LNPs in which mRNA activity was detected in multiple organs; these LNPs are plotted in the region of the organ for which higher activity was measured. For the 67 LNPs tested with the TNS assay, LNP apparent pK_a was correlated with the specificity of luciferase mRNA tissue delivery.Herein, we identify and study mechanistic factors which could explain the organ-targeting properties of SORT LNPs based on three established principles that define the efficacy of liver-targeting LNPs: biodistribution to the liver, an acid dissociation constant (pK_a) near 6.4, and the adsorption of apolipoprotein E (ApoE) to the LNP surface (12, 28). We discovered that each of these three factors (organ level biodistribution, apparent pK_a, and serum protein adsorption) are distinct for SORT LNPs and correlate with their tissue-targeting properties. Furthermore, we provide evidence of a three-step mechanism for the functional role of serum protein adsorption on tissue targeting that extends the scope and application of the endogenous targeting mechanism defined for four-component LNP delivery to liver hepatocytes (12, 28). First, desorption of PEG lipids on the LNP surface exposes underlying SORT molecules in the LNP. Next, distinct serum proteins recognize the exposed SORT molecules and adsorb to the LNP surface. Finally, surface-adsorbed proteins interact with cognate receptors expressed by cells in the target organs to facilitate functional mRNA delivery to those tissues.The results indicate that the choice of SORT molecule governs which proteins most avidly adsorb to the LNP surface, impacting the ultimate biological fate of the LNP (29). We envision that this mechanistic understanding of SORT LNPs will enable their optimization for therapeutic applications in the lung, liver, and spleen and lay the foundation for extending the SORT platform to additional nanoparticle types, physiological tissues, and cell types. Furthermore, our findings suggest that endogenous targeting—that is, tuning a nanoparticle’s molecular composition such that it binds specific proteins in the serum to enable delivery to the target site—could be a generally useful strategy for engineering a wide array of nanomaterials capable of extrahepatic delivery.     

Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus

Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production.     

Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.     

Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.     

Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3

Loss-of-function mutations in Angiopoietin-like 3 (Angptl3) are associated with lowered blood lipid levels, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders. In this study, we developed a lipid nanoparticle delivery platform carrying Cas9 messenger RNA (mRNA) and guide RNA for CRISPR-Cas9–based genome editing of Angptl3 in vivo. This system mediated specific and efficient Angptl3 gene knockdown in the liver of wild-type C57BL/6 mice, resulting in profound reductions in serum ANGPTL3 protein, low density lipoprotein cholesterol, and triglyceride levels. Our delivery platform is significantly more efficient than the FDA-approved MC-3 LNP, the current gold standard. No evidence of off-target mutagenesis was detected at any of the nine top-predicted sites, and no evidence of toxicity was detected in the liver. Importantly, the therapeutic effect of genome editing was stable for at least 100 d after a single dose administration. This study highlights the potential of LNP-mediated delivery as a specific, effective, and safe platform for Cas9-based therapeutics.

Genome engineering has recently emerged as a potentially powerful therapeutic tool for the treatment of diseases with a genetic etiology. For monogenic disorders, the mutated locus can be directly targeted for repair via genome editing. For complex polygenic disorders such as hyperlipidemia, which are the result of a combination of the effects of a large number of genes as well as nutritional and environmental influences, identifying a single gene to repair may be challenging. An alternative therapeutic approach has been to identify monogenic mutations found naturally in the human population that confer a protection against the disease. By introducing these protective loci, it may be possible to develop a single therapeutic that is effective for many patients, regardless of their specific genetic backgrounds. This strategy has proven successful in the case of Pcsk9, wherein loss of function protects against hyperlipidemia: small interfering RNA (siRNA)-mediated Pcsk9 knockdown strategies have recently completed Phase III clinical trials for the treatment of hyperlipidemia (1, 2).Angiopoietin-like 3 (ANGPTL3) is an enzyme which regulates plasma lipoprotein levels (3). ANGPTL3 deficiency exists naturally in the human population, resulting from loss-of-function mutations in the Angptl3 gene (4). These individuals show lowered blood triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C), without any apparent clinical risks or complications resulting from this loss (5, 6). Recent genetic and pharmacologic studies have validated this finding and indicated that Angptl3 knockdown may confer some protective benefits, making Angptl3 an attractive therapeutic target for the treatment of human lipoprotein metabolism disorders (7, 8). Two different therapeutic inhibition strategies against ANGPTL3 have recently been validated. In one clinical trial, an ANGPTL3-targeting monoclonal antibody, evinacumab, has proven effective for reducing LDL-C and TG levels in healthy human volunteers (8, 9). These results are in line with a study that found administration of an antisense oligonucleotide (ASO) targeting Angptl3 messenger RNA (mRNA) achieved reduced lipid levels as well as decreased progression of atherosclerosis in mice (10). Furthermore, lowered levels of atherogenic lipoproteins in humans was also observed, and no serious adverse events were documented in the Phase I randomized clinical trial (10). These observations suggest that therapeutic antagonism of ANGPTL3 is effective and safe for reducing levels of lipids and incidences of atherosclerotic cardiovascular disease.The microbial CRISPR-Cas9 system has been developed as a genome editing tool (11, 12). Cas9 introduces DNA double-strand breaks (DSBs) at sites targeted by a complementary guide RNA (gRNA). These DSBs are then repaired either by nonhomologous end joining or homology directed repair (13, 14). As compared with conventional ASO or antibody therapies, which are transient, the CRISPR-Cas9 system can induce permanent loss-of-function mutations, resulting in long-term therapeutic effects in the edited cells. However, the safe, efficient, and specific delivery of CRISPR-based therapies is a significant technical challenge, which has limited the therapeutic application of this technology. Viral vectors (such as adenovirus or adeno-associated virus) have been used for delivery of CRISPR-based therapies, including editing Angptl3 (15), and while they can lead to high editing efficiency, they also carry significant safety risks associated with undesired insertional mutagenesis and potential biosafety (16, 17). An alternative, promising delivery modality is nonviral nanoparticles, such as lipid nanoparticles (LNPs), gold nanoparticles, or polymeric nanoparticles. LNPs have an advantageous safety profile and have been developed for the delivery of Cas9 plasmid DNA, mRNA, and ribonucleoproteins (RNPs), albeit with reduced delivery efficiency compared to viral vector approaches (18–20). We and others have previously reported using LNPs to successfully deliver CRISPR-Cas9 in both the RNP and mRNA formats (21–25). While delivering Cas9 as DNA, mRNA, or RNP (with gRNA) formats each have potential strengths, mRNA delivery is particularly promising for in vivo genome editing applications because of its transient, nonintegrating Cas9 expression feature.Recently, LNPs have been explored by us and others for the delivery of Cas9 mRNA in vitro and in vivo. We recently demonstrated the use of bioreducible LNPs for the codelivery of Cas9 mRNA and gRNA, demonstrating highly efficient in vitro genome editing, as well as rapid knockdown of the Pcsk9 cholesterol-regulating gene in vivo, highlighting the potential of this delivery approach (26). However, our previous study did not thoroughly examine the long-term therapeutic effects of this treatment. Recent research by Finn et al. (22) examined the ability to modulate the chemical structure of the Cas9 gRNA to influence the Cas9 editing efficiency in vivo to knock down the Ttr gene, a monogenic target overexpressed in certain orphan-designated amyloidosis diseases (22).Here, we describe a highly potent nonviral LNP-mediated CRISPR-Cas9 delivery system for the liver delivery of Cas9 mRNA and demonstrate its efficacy by targeting the Angptl3 gene. The system is composed of a leading tail-branched bioreducible lipidoid (306-O12B) coformulated with an optimized mixture of excipient lipid molecules, and it successfully codelivers SpCas9 mRNA and a single-guide RNA (sgRNA) targeting Angptl3 (sgAngptl3) via a single administration (Fig. 1). 306-O12B LNP specifically delivered Cas9 mRNA and sgAngptl3 to liver hepatocytes of wild-type C57BL/6 mice, resulting in a median editing rate of 38.5% and a corresponding 65.2% reduction of serum ANGPTL3 protein. We found that delivery using 306-O12B LNP was more efficient than delivery with MC-3 LNP, a gold standard LNP that was recently approved by the US Food and Drug Administration (FDA) for liver-targeted delivery of nucleic acids (27). Moreover, liver specific knockdown of Angptl3 resulted in profound lowering of LDL-C and TG levels. Importantly, no evidence of off-target mutagenesis at nine top-predicted sites was observed nor any apparent liver toxicity. The CRISPR-mediated genome editing was maintained at a therapeutically relevant level for at least 100 d after the injection of a single dose. The system we established here offers a clinically viable approach for liver-specific delivery of CRISPR-Cas9–based genome editing tools.Open in a separate windowFig. 1.Schematic illustration of LNP-mediated in vivo CRISPR-Cas9–based genome editing to induce loss-of-function mutations in Angptl3 to lower blood lipid levels. The Cas9 mRNA and Angptl3-specific sgRNA (sgAngptl3) are encapsulated in the LNP and delivered to the liver hepatocytes where they cleave the Angptl3 target locus, leading to reduced ANGPTL3 protein. Reduced ANGPTL3 level leads to a disinhibition of Lipoprotein lipase (LPL), which allows LPL to regulate the levels of therapeutically relevant circulating lipids such as LDL-C and TG.     

Modeling suggests gene editing combined with vaccination could eliminate a persistent disease in livestock

Recent breakthroughs in gene-editing technologies that can render individual animals fully resistant to infections may offer unprecedented opportunities for controlling future epidemics in farm animals. Yet, their potential for reducing disease spread is poorly understood as the necessary theoretical framework for estimating epidemiological effects arising from gene-editing applications is currently lacking. Here, we develop semistochastic modeling approaches to investigate how the adoption of gene editing may affect infectious disease prevalence in farmed animal populations and the prospects and time scale for disease elimination. We apply our models to the porcine reproductive and respiratory syndrome (PRRS), one of the most persistent global livestock diseases to date. Whereas extensive control efforts have shown limited success, recent production of gene-edited pigs that are fully resistant to the PRRS virus have raised expectations for eliminating this deadly disease. Our models predict that disease elimination on a national scale would be difficult to achieve if gene editing was used as the only disease control. However, from a purely epidemiological perspective, disease elimination may be achievable within 3 to 6 y, if gene editing were complemented with widespread and sufficiently effective vaccination. Besides strategic distribution of genetically resistant animals, several other key determinants underpinning the epidemiological impact of gene editing were identified.

Novel genomic technologies such as gene editing offer promising opportunities to tackle some of the most pressing global challenges humanity faces today. They provide new prospects to solving emerging threats such as the global COVID-19 pandemic (1) as well as to long-standing global health issues such as the HIV/AIDS crisis (2) or malnutrition (3, 4), with minimal side effects. Besides the medical field, food production stands to gain most from widespread use of genome editing technologies. Currently 11% of the human population suffers malnourishment (5), and this is expected to increase with the projected growth of the human population to 10.9 billion by 2100 (42%) (6). Meeting the 60% increase of agricultural production needed to provide sustainable and nutritious diets will likely require transformative innovations to existing production methods (7). While genome-editing technologies have been applied widely in plant breeding to simultaneously improve production and resilience to diverse stressors (see ref. 8 for examples), their application in the livestock sector is still in its infancy, primarily due to technical limitations associated with the gene-editing process itself and the safe and fast dissemination of edits, as well as ethical and societal concerns (9). Nevertheless, breakthroughs in genetic modification of farm animals through genome editing start to emerge with drastic improvements in efficiency traits (10, 11), animal welfare (12), and disease resistance (13, 14). Improving disease resistance in livestock seems particularly pertinent, as infectious diseases affect the entire food production chain and its economic viability (15).The recent scientific breakthroughs in genome editing raise expectations for radical shifts in infectious disease control in livestock (14). Although many countries currently lack specific regulations covering the application of genome-edited animals in the food chain, this technology currently falls under genetically modified organism legislation in countries that have such processes. Reflecting this, we are seeing the rapid development of gene-editing regulations worldwide [see the Global Gene Editing Regulation Tracker (16) for an up-to-date status of gene-editing regulations per country]. Specifically, some countries have identified that some genome-editing strategies are exempt from regulatory approval. This is reflected in the recent announcement in Japan that a genome-edited seabream does not need to be regulated as no gene has been introduced into the genome (17, 18). These developments make it realistic that application of gene editing to help control infectious disease is likely in the near future. This prospect evokes pressing questions concerning the theoretical and practical feasibility of tackling diseases for which conventional control methods have failed. It is currently not known how to best implement gene-editing-induced disease resistance to achieve noticeable reduction in disease prevalence and possibly even eliminate the disease on a national scale, and on what time scale such ambitious goals could be achieved.These questions are impossible to address in an entirely hypothetical context since epidemiological characteristics affecting the spread of the disease in question and the dynamics of the dispersal of resistant animals within the population play important roles in the success of the scheme. In this study, we focus on a particular disease, porcine reproductive and respiratory syndrome (PRRS), for the development of a mathematical modeling framework to investigate the feasibility of the application of gene editing to achieve disease elimination. PRRS represents one of the most important infectious disease problems for the pig industry worldwide, with economic losses estimated at $2.5 billion per annum in the United States and Europe alone (19, 20). Despite extensive global control efforts, the disease continues to persist in national commercial pig populations, largely due to high genetic heterogeneity of the PRRS virus, PRRSV (21), and the associated limited effectiveness of all PRRS vaccines (22, 23) and limited reliability of diagnostic tests (24, 25). There is considerable natural genetic variation in pigs’ responses to PRRSV infection, but evidence to date suggests that no pig strain is naturally fully resistant to it (26). However, recent advances in gene editing of porcine macrophages, in which a simple disruption of the CD163 gene confers complete resistance to infection with PRRSV, may revolutionize future PRRS control (27–29).To exploit the full potential of gene editing for PRRS control, we here develop a theoretical proof-of-concept model to address a number of crucial research questions. To what extent can gene editing help reduce PRRS prevalence in national commercial pig populations? Is it possible to eliminate this disease through gene editing by creating a disease-resistant subpopulation adequately dispersed within the national susceptible population? If so, what proportion of pigs would have to be PRRSV-resistant and how would these animals need to be distributed across herds?It is unlikely that gene editing will fully replace existing control methods, such as widespread vaccination. Hence, we also use our model to investigate the epidemiological effects of gene editing and vaccination combined. Finally, we investigate how fast the required proportion of resistant animals could be introduced in a national commercial pig population, if gene editing was strictly limited to breeding programs and resistance alleles propagate to commercial pigs using current industry practices with their diverse technical limitations. This last question becomes particularly important for an RNA virus with a high evolutionary rate such as PRRSV, since escape mutants of the virus might limit the shelf-life of gene editing and vaccines in terms of effectiveness (14, 30).We address these questions with two linked simulation models: 1) an epidemiological model to simulate the effects of different disease control schemes on PRRS prevalence in a national commercial pig population and 2) a gene flow model to simulate the propagation of PRRSV resistance alleles from breeding programs that routinely carry out gene editing for PRRS resistance into the commercial population. The epidemiological model provides insight into the numbers and distribution of genetically resistant pigs required to eliminate PRRS under a range of realistic scenarios. The allele propagation model subsequently provides estimates for the time required to realistically produce this required number of genetically resistant pigs.Our proof-of-concept model provides quantitative estimates for how gene editing may reduce infectious disease prevalence in farm animals and the required time frame and criteria for eliminating a disease on a national level.     

用CRISPR/Cas9系统消除潜伏的HIV前病毒基因组及其感染细胞的研究进展

艾滋病在全球采用联合抗逆转录病毒治疗后发病率及死亡率呈持续下降趋势,使之成为一种可管理的慢性传染病。但因受各种因素制约,艾滋病仍然是全球一个重要公共卫生问题。HIV/AIDS持续存在的主要原因是现有的治疗无法清除人体中存在的HIV潜伏库,由于这种库的存在,HIV/AIDS患者必须终生使用抗病毒药物来抑制病毒复制和反弹。成簇规律间隔短回文重复序列和相关核酸酶Cas9(CRISPR/Cas9)系统几年前以一种简单、快速及易操作的基因编辑技术问世,多项研究表明其在HIV感染的细胞和在动物模型实验中具有消除或破坏HIV储存库或HIV感染细胞的潜力,可能由此产生治愈HIV/AIDS的方法。本文分析了CRISPR/CAS9系统应用于消除潜伏HIV的结果,并对可能产生的问题和趋势进行了讨论。     

Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography–mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

The use of messenger RNA (mRNA) for vaccination (1, 2), protein replacement therapy (3) and cancer immunotherapy (4), and mRNA technology encoding CRISPR/Cas nuclease for genome editing (5) holds the potential to revolutionize the treatment of a wide range of currently untreatable genetic diseases. The US Food and Drug Administration (FDA) recently authorized two mRNA vaccines enabled by nonviral lipid nanoparticles (LNPs) against COVID-19 for emergency use, representing a key milestone in mRNA therapeutics. Aside from COVID-19, other mRNA vaccines against influenza viruses (6), Cytomegalovirus (7), and advanced melanoma (8) have also been developed and are now in human clinical trials. The clinical success of these transformative therapeutics is largely reliant on the development of safe, efficient, and highly selective delivery systems to target mRNA toward specific tissues and cell types (9, 10).As one of the most advanced nonviral synthetic nanoparticles, LNPs have been proven to specifically deliver small interfering RNA (siRNA) to the liver for the treatment of hereditary transthyretin amyloidosis (11). Since mRNA predominantly accumulates in the liver and spleen following systemic delivery (12–16), much of the clinical interest to date has focused on hepatic diseases. Delivery vehicles that enable specific mRNA delivery to extrahepatic tissues are urgently needed to fully realize the potential of mRNA-based therapy. Considerable effort has been made to develop organ-targeted LNPs to bypass liver accumulation by modifying the surface of LNPs with targeting moieties such as peptides, antibodies, and proteins (17–19). Recently, targeted LNPs functionalized with alpha plasmalemma vesicle–associated protein antibody were developed for lung-targeted mRNA delivery in vivo (18). More recently, a selective organ targeting (SORT) strategy was developed to engineer LNPs to tune the biodistribution of LNPs; the incorporation of an extra excipient, the SORT molecule, can enable the precise alteration of the in vivo mRNA delivery profile (20). These strategies exhibit advantages in mitigating liver accumulation and delivering mRNA to lungs or spleens. These promising developments motivate us to continue explore innovative ways to deliver mRNA to specific locations.A major roadblock in the development of targeted LNPs is difficulty predicting the in vivo targeting behavior of newly designed LNPs due to the limited understanding of the nano-bio interactions between nanoparticles (NPs) and biological components. The outer surface of NPs can be rapidly covered with a layer of serum proteins, referred to as the “protein corona,” which remodels the surface property of NPs and substantially affects the interaction of NPs with organs and cells (21). We and others have demonstrated that the lipidoid amine head structure can impact the delivery efficacy and even the in vivo targetability of mRNA-loaded LNPs (22–24). In a recent study, we showed that imidazole-based synthetic lipidoids preferentially target mRNA to the spleen (25). For the lipidoid tail chemistry, although considerable progress has been made in the understanding of lipidoid tail length, degree of unsaturation, and degree of branching on the effect of mRNA delivery potency (26–29), the influence of lipidoid tail structures on the in vivo selectivity of LNPs remains poorly understood. To address this important knowledge gap, we synthesized a library of amide bond–containing lipidoids (N-series LNPs) via Michael addition reaction between amine heads and acrylamide tails (Fig. 1A). Surprisingly, from in vivo screening, we found that the N-series LNPs almost exclusively deliver mRNA to the lung following systemic administration (Fig. 1 B and C). Intriguingly, our previous study demonstrated that the O-series lipidoids, which contain an ester bond in the tails, tend to deliver mRNA into the liver (16). To better understand why such a small change induces such striking organ specificity, we further investigated the underlying mechanisms of these delivery differences. We hypothesized that once injected into the bloodstream, the LNPs can selectively govern the adsorption of specific plasma proteins to serve as targeting ligands that direct LNPs to selected organs. Indeed, using proteomics, we identified a group of unique plasma proteins specifically absorbed on the surface of two representative LNP candidates, 306-O12B and 306-N16B, that may affect the targetability of these LNPs. More importantly, we found that different pulmonary subcellular populations can be targeted by changing the lipidoid head structure of N-series LNPs. Furthermore, we evaluated the lung-targeting LNPs for the in vivo targeted delivery of Tsc2 mRNA to TSC2-deficient cells to restore the expression of the TSC2 tumor suppressor for the treatment of pulmonary lymphangioleiomyomatosis (LAM), a rare genetic disorder caused by biallelic mutations and loss of function of TSC complex genes. This study provides proof of concept that tuning the in vivo organ-targeting behavior of LNPs can be achieved by tailoring the composition of protein corona via simple chemistry. This work provides a strategy for the rational design of highly specific organ- and cell-selective LNPs for mRNA-based therapy.Open in a separate windowFig. 1.Synthesis and in vivo screening of N-series LNPs. (A) Synthetic route and representative chemical structure of lipidoids. Representative whole-body bioluminescence images of mice (B) and in vivo mRNA delivery efficacy (C) of N-series LNPs measured by the IVIS imaging system. Mice were injected with either of the Luc mRNA–loaded N-series LNPs at a single dose of 0.5 mg/kg. Images were taken at 6 h postinjection (n = 3). Data are presented as mean ± SD; the error bar around each data point is the SEM.     

Plant Viruses: From Targets to Tools for CRISPR

Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for ‘clustered regularly interspaced short palindromic repeats’ and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.     

Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.

Eukaryotic ribosomes are highly complex protein synthesis machinery composed of the small/40S subunit and the large/60S subunit. Historically, the ribosome was considered to be an invariant effector rather than a regulatory participant in translation. However, emerging studies have revealed heterogeneity among ribosomes, characterized by variable ribosomal protein (RP) contents and consequent specialization of the translational program for modulating growth robustness and stress response (1–4). Such findings led to the formulation of the concept of “specialized ribosomes”, wherein multiple populations of ribosomes with diverse compositions were produced, and each was tailored to carry different translational abilities, such as mRNA selectivity during translation as well as translation fidelity and elongation speed (5–7). Although current evidence proves the existence of ribosome heterogeneity and the functional specialization for distinct ribosomes in selectively translating specific subsets of messenger RNAs (mRNAs), the sources of ribosome heterogeneity and thereafter the output remain largely unknown and are subjects of great interest (5–7).In both lower and higher eukaryotes, many RPs are encoded by paralogous genes, possibly originating from genome duplication early in evolution and maintained afterward (7–9). For example, 24 of the 33 RPs in the yeast 40S ribosome subunit, which contains the ribosome decoding center, exist as paralog pairs, which allows 2²⁴ (>10⁷) potential RP combinations by numbers alone. To date, numerous studies have examined the roles of individual RP paralogs in diverse organisms (e.g., yeast, amoebas, flies, and vertebrates), and, accordingly, rapidly growing evidence has shown that RP paralog pairs usually possess distinct or even opposite expression patterns and paralog-specific functions (10–12). Thus, heterogeneous RP paralogs not only regulate cell function by complementing each other’s expression levels (dose amplification) but also offer the potential to generate heterogeneous ribosomes by swapping distinct RP isoforms to fine-tune the translational program (paralog-specific regulation) (2, 10–12). However, direct evaluation of their effects on cell function, especially the functionalities of ribosomes, was obscured by the highly diversified cellular RP pool (5, 6).In this study, we homogenized the cellular 40S RP pool by removing one set of paralogous genes and consequently constructed a “designer” yeast named homo-40S. Although the successful engineering of homo-40S demonstrated that the presence of duplicated RP paralogs was not essential for yeast survival, our results showed that loss of RP paralogs in homo-40S led to decreased fitness and increased susceptibility to the translation error-inducing inhibitor paromomycin, which is indicative of altered translational activity and fidelity. Moreover, these phenotypes could be recovered markedly, although not fully, by doubling the RP paralogous genes in homo-40S. Furthermore, our study showed that homo-40S was able to improve paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to acquire fast-growing derivatives. Together, our work provides a strategy to acquire monotypic ribosomes with homogenized RP contents and experimentally demonstrates the functional significance for maintaining duplicated RP genes.