Targeted genome-modification tools and their advanced applications in crop breeding.

Crop improvement by genome editing involves the targeted alteration of genes to improve plant traits, such as stress tolerance, disease resistance or nutritional content. Techniques for the targeted modification of genomes have evolved from generating random mutations to precise base substitutions, followed by insertions, substitutions and deletions of small DNA fragments, and are finally starting to achieve precision manipulation of large DNA segments. Recent developments in base editing, prime editing and other CRISPR-associated systems have laid a solid technological foundation to enable plant basic research and precise molecular breeding. In this Review, we systematically outline the technological principles underlying precise and targeted genome-modification methods. We also review methods for the delivery of genome-editing reagents in plants and outline emerging crop-breeding strategies based on targeted genome modification. Finally, we consider potential future developments in precise genome-editing technologies, delivery methods and crop-breeding approaches, as well as regulatory policies for genome-editing products.

Anlotinib enhanced CD8+ T cell infiltration via induction of CCL5 improves the efficacy of PD-1/PD-L1 blockade therapy in lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of mortality worldwide and requires effective treatment strategies. Recently, the development of a novel multiple-target tyrosine kinase inhibitor, anlotinib, has drawn increasing attention, especially it shows advantages when combined with PD-1/PD-L1 blockade. However, the mechanism by which anlotinib improves immunotherapy and remodeling of the tumor microenvironment remains unclear. In this study, we found that anlotinib combined with PD-1 blockade significantly inhibited tumor growth and reduced tumor weight in a lung cancer xenograft model compared to any single treatment. Both immunofluorescence and flow cytometry analyses revealed that anlotinib induced a CD8+ T cell dominated tumor microenvironment, which might account for its improved role in immunotherapy. Further investigations showed that CCL5-mediated CD8+ T cell recruitment plays a critical role in anlotinib and PD-1 blockade strategies. The depletion of CD8+ T cells abrogated this process. In conclusion, our findings showed that the combination of anlotinib and PD-1 blockade produced promising effects in the treatment of lung cancer, and that the induction of CCL5-mediced CD8+ T cell recruitment by anlotinib provided a novel mechanism of action.

Hypoxia-Derived Exosomes Promote Lung Adenocarcinoma by Regulating HS3ST1-GPC4-Mediated Glycolysis.

OBJECTIVE: The diagnosis of lung adenocarcinoma (LUAD) is often delayed due to the typically asymptomatic nature of the early-stage disease, causing advanced-stage LUAD diagnosis in most patients. Hypoxia is widely recognized as a driving force in cancer progression. Exosomes originating from hypoxic tumor cells promote tumorigenesis by influencing glycolysis, migration, invasion, and immune infiltration. Given these insights, our study aimed to explore the role of hypoxia-derived exosomal long non-coding RNA (lncRNA) OIP5-AS1 in LUAD cell lines and mouse models. MATERIALS AND METHODS: Exosomes were meticulously isolated and authenticated based on their morphology and biomarkers. The interaction between heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) and Glypican 4 (GPC4) was examined using immunoprecipitation. The influence of the hypoxia-derived exosomal lncRNA OIP5-AS1 on glycolysis was assessed in LUAD cell lines. The effect of the hypoxia-derived exosomal lncRNA OIP5-AS1 on cell proliferation and metastasis was evaluated using colony formation, cell viability, cell cycle, and apoptosis analyses. Its effects on tumor size were confirmed in xenograft animal models. RESULTS: Our study revealed the mechanism of the hypoxia-derived exosomal lncRNA OIP5-AS1 in LUAD progression. We discovered that GPC4 promotes HS3ST1-mediated glycolysis and that the hypoxia-derived exosomal lncRNA OIP5-AS1 enhances glycolysis by regulating miR-200c-3p in LUAD cells. Notably, this lncRNA stimulates LUAD cell proliferation and metastasis and fosters LUAD tumor size via miR-200c-3p. Our findings underscore the potential role of the hypoxia-derived exosomal lncRNA OIP5-AS1 in LUAD progression. CONCLUSIONS: The hypoxia-derived exosomal lncRNA OIP5-AS1 promotes LUAD by regulating HS3ST1-GPC4-mediated glycolysis via miR-200c-3p.

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

Identification of the genetic characteristics of copy number variations in experimental specific pathogen-free ducks using whole-genome resequencing.

BACKGROUND: Specific pathogen-free ducks are a valuable laboratory resource for waterfowl disease research and poultry vaccine development. High throughput sequencing allows the systematic identification of structural variants in genomes. Copy number variation (CNV) can explain the variation of important duck genetic traits. Herein, the genome-wide CNVs of the three experimental duck species in China (Jinding ducks (JD), Shaoxing ducks (SX), and Fujian Shanma ducks (SM)) were characterized using resequencing to determine their genetic characteristics and selection signatures. RESULTS: We obtained 4,810 CNV regions (CNVRs) by merging 73,012 CNVs, covering 4.2% of the duck genome. Functional analysis revealed that the shared CNVR-harbored genes were significantly enriched for 31 gene ontology terms and 16 Kyoto Encyclopedia of Genes and Genomes pathways (e.g., olfactory transduction and immune system). Based on the genome-wide fixation index for each CNVR, growth (SPAG17 and PTH1R), disease resistance (CATHL3 and DMBT1), and thermoregulation (TRPC4 and SLIT3) candidate genes were identified in strongly selected signatures specific to JD, SM, and SX, respectively. CONCLUSIONS: In conclusion, we investigated the genome-wide distribution of experimental duck CNVs, providing a reference to establish the genetic basis of different phenotypic traits, thus contributing to the management of experimental animal genetic resources.

Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.

Precise integration of large DNA sequences in plant genomes using PrimeRoot editors.

A technique for chromosomal insertion of large DNA segments is much needed in plant breeding and synthetic biology to facilitate the introduction of desired agronomic traits and signaling and metabolic pathways. Here we describe PrimeRoot, a genome editing approach to generate targeted precise large DNA insertions in plants. Third-generation PrimeRoot editors employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genomes. We demonstrate the use of PrimeRoot to accurately introduce gene regulatory elements in rice. In this study, we also integrated a gene cassette comprising PigmR, which confers rice blast resistance driven by an Act1 promoter, into a predicted genomic safe harbor site of Kitaake rice and obtain edited plants harboring the expected insertion with an efficiency of 6.3%. We found that these rice plants have increased blast resistance. These results establish PrimeRoot as a promising approach to precisely insert large segments of DNA in plants.

Epigenetic modifications regulate cultivar-specific root development and metabolic adaptation to nitrogen availability in wheat.

The breeding of crops with improved nitrogen use efficiency (NUE) is crucial for sustainable agriculture, but the involvement of epigenetic modifications remains unexplored. Here, we analyze the chromatin landscapes of two wheat cultivars (KN9204 and J411) that differ in NUE under varied nitrogen conditions. The expression of nitrogen metabolism genes is closely linked to variation in histone modification instead of differences in DNA sequence. Epigenetic modifications exhibit clear cultivar-specificity, which likely contributes to distinct agronomic traits. Additionally, low nitrogen (LN) induces H3K27ac and H3K27me3 to significantly enhance root growth in KN9204, while remarkably inducing NRT2 in J411. Evidence from histone deacetylase inhibitor treatment and transgenic plants with loss function of H3K27me3 methyltransferase shows that changes in epigenetic modifications could alter the strategy preference for root development or nitrogen uptake in response to LN. Here, we show the importance of epigenetic regulation in mediating cultivar-specific adaptation to LN in wheat.

Strand-preferred base editing of organellar and nuclear genomes using CyDENT.

Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination. We demonstrate effective base editing in nuclear, mitochondrial and chloroplast genomes. At certain mitochondrial sites, we show editing efficiencies of 14% and strand specificity of 95%. Furthermore, by exchanging the CyDENT deaminase with one that prefers editing GC motifs, we demonstrate up to 20% mitochondrial base editing at sites that are otherwise inaccessible to editing by other methods. The modular nature of CyDENT enables a suite of bespoke base editors for various applications.

Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors.

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Whole-genome resequencing to unveil genetic characteristics and selection signatures of specific pathogen-free ducks.

Specific pathogen-free ducks are important high-grade laboratory animals, with a key role in research related to poultry biosecurity, production, and breeding. However, the genetic characteristics of experimental duck varieties remain poorly explored. Herein we performed whole-genome resequencing to construct a single nucleotide polymorphism genetic map of the genomes of 3 experimental duck varieties [Jinding ducks (JD), Shaoxing ducks (SX), and Fujian Shanma ducks (SM)] to determine their genetic characteristics and identify selection signatures. Subsequent analyses of population structure and genetic diversity revealed that each duck variety formed a monophyletic group, with SM showing richer genetic diversity than JD and SX. Further, on exploring shared selection signatures, we found 2 overlapping genomic regions on chromosome Z of all experimental ducks, which comprised immune response-related genes (IL7R and IL6ST). Moreover, growth and skeletal development (IGF1R and GDF5), meat quality (FoxO1), and stress resistance (HSP90B1 and Gpx8-b) candidate gene loci were identified in strongly selected signatures specific to JD, SM, and SX, respectively. Our results identified the population genetic basis of experimental ducks at the whole-genome level, providing a framework for future molecular investigations of genetic variations and phenotypic changes. We believe that such studies will eventually contribute to the management of experimental animal resources.

Tuning plant phenotypes by precise, graded downregulation of gene expression.

The ability to control gene expression and generate quantitative phenotypic changes is essential for breeding new and desired traits into crops. Here we report an efficient, facile method for downregulating gene expression to predictable, desired levels by engineering upstream open reading frames (uORFs). We used base editing or prime editing to generate de novo uORFs or to extend existing uORFs by mutating their stop codons. By combining these approaches, we generated a suite of uORFs that incrementally downregulate the translation of primary open reading frames (pORFs) to 2.5-84.9% of the wild-type level. By editing the 5' untranslated region of OsDLT, which encodes a member of the GRAS family and is involved in the brassinosteroid transduction pathway, we obtained, as predicted, a series of rice plants with varied plant heights and tiller numbers. These methods offer an efficient way to obtain genome-edited plants with graded expression of traits.

Applying T-classifier, binary classifiers, upon high-throughput TCR sequencing output to identify cytomegalovirus exposure history.

With the continuous development of information technology and the running speed of computers, the development of informatization has led to the generation of increasingly more medical data. Solving unmet needs such as employing the constantly developing artificial intelligence technology to medical data and providing support for the medical industry is a hot research topic. Cytomegalovirus (CMV) is a kind of virus that exists widely in nature with strict species specificity, and the infection rate among Chinese adults is more than 95%. Therefore, the detection of CMV is of great importance since the vast majority of infected patients are in a state of invisible infection after the infection, except for a few patients with clinical symptoms. In this study, we present a new method to detect CMV infection status by analyzing high-throughput sequencing results of T cell receptor beta chains (TCRß). Based on the high-throughput sequencing data of 640 subjects from cohort 1, Fisher's exact test was performed to evaluate the relationship between TCRß sequences and CMV status. Furthermore, the number of subjects with these correlated sequences to different degrees in cohort 1 and cohort 2 were measured to build binary classifier models to identify whether the subject was CMV positive or negative. We select four binary classification algorithms: logistic regression (LR), support vector machine (SVM), random forest (RF), and linear discriminant analysis (LDA) for side-by-side comparison. According to the performance of different algorithms corresponding to different thresholds, four optimal binary classification algorithm models are obtained. The logistic regression algorithm performs best when Fisher's exact test threshold is 10-5, and the sensitivity and specificity are 87.5% and 96.88%, respectively. The RF algorithm performs better at the threshold of 10-5, with a sensitivity of 87.5% and a specificity of 90.63%. The SVM algorithm also achieves high accuracy at the threshold value of 10-5, with a sensitivity of 85.42% and specificity of 96.88%. The LDA algorithm achieves high accuracy with 95.83% sensitivity and 90.63% specificity when the threshold value is 10-4. This is probably because the two-dimensional distribution of CMV data samples is linearly separable, and linear division models such as LDA are more effective, while the division effect of nonlinear separable algorithms such as random forest is relatively inaccurate. This new finding may be a potential diagnostic method for CMV and may even be applicable to other viruses, such as the infectious history detection of the new coronavirus.

Enabling technology and core theory of synthetic biology.

Synthetic biology provides a new paradigm for life science research ("build to learn") and opens the future journey of biotechnology ("build to use"). Here, we discuss advances of various principles and technologies in the mainstream of the enabling technology of synthetic biology, including synthesis and assembly of a genome, DNA storage, gene editing, molecular evolution and de novo design of function proteins, cell and gene circuit engineering, cell-free synthetic biology, artificial intelligence (AI)-aided synthetic biology, as well as biofoundries. We also introduce the concept of quantitative synthetic biology, which is guiding synthetic biology towards increased accuracy and predictability or the real rational design. We conclude that synthetic biology will establish its disciplinary system with the iterative development of enabling technologies and the maturity of the core theory.

Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs).

Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months.