All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Engineering a Cortisol Sensing Enteric Probiotic.

Chronic stress can lead to prolonged adrenal gland secretion of cortisol, resulting in human ailments such as anxiety, post-traumatic stress disorder, metabolic syndrome, diabetes, immunosuppression, and cardiomyopathy. Real time monitoring of chronic increases in cortisol and intervening therapies to minimize the physiological effects of stress would be beneficial to prevent these endocrine related illnesses. Gut microbiota have shown the ability to secrete, respond, and even regulate endocrine hormones. One such microbe, Clostridium scindens, responds transcriptionally to cortisol. We engineered these cortisol responsive genetic elements from C. scindens into an enteric probiotic, E. coli Nissle 1917, to drive the expression of a fluorescent reporter allowing for the designing, testing, and building of a robust and physiologically relevant novel cortisol probiotic sensor. This smart probiotic was further engineered to be more sensitive and to respond to elevated cortisol by expressing tryptophan decarboxylase, thereby bestowing the ability to generate tryptamine and serotonin. Here we show that upon cortisol treatment the smart probiotic produces measurable amounts of tryptamine. Accumulated levels of these neuromodulators should improve mood, anxiety, and depression and drive down cortisol levels. Importantly, this work can serve as a model for the engineering of a sense-and-respond probiotic to modulate the gut-brain axis.

The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida.

The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at attTn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique 'landing pads' for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of rrn operons. Integration in the attTn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/PnagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.

A landing pad system for multicopy gene integration in Issatchenkia orientalis.

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user's choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

A splinkerette PCR-based genome walking technique for the identification of transgene integration sites in CHO cells.

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica.

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.

Bacterial DNA involvement in carcinogenesis.

The incidence of cancer is high worldwide, and biological factors such as viruses and bacteria play an important role in the occurrence of cancer. Helicobacter pylori, human papillomavirus, hepatitis B viruses and other organisms have been identified as carcinogens. Cancer is a disease driven by the accumulation of genome changes. Viruses can directly cause cancer by changing the genetic composition of the human body, such as cervical cancer caused by human papillomavirus DNA integration and liver cancer caused by hepatitis B virus DNA integration. Recently, bacterial DNA has been found around cancers such as pancreatic cancer, breast cancer and colorectal cancer, and the idea that bacterial genes can also be integrated into the human genome has become a hot topic. In the present paper, we reviewed the latest phenomenon and specific integration mechanism of bacterial DNA into the human genome. Based on these findings, we also suggest three sources of bacterial DNA in cancers: bacterial DNA around human tissues, free bacterial DNA in bacteremia or sepsis, and endogenous bacterial DNA in the human genome. Clarifying the theory that bacterial DNA integrates into the human genome can provide a new perspective for cancer prevention and treatment.

Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae.

Construction of efficient microbial cell factories always requires assembling biosynthetic pathways and rewiring cellular metabolism with overexpression of multiple genes. Genomic integration is considered to be helpful for stable gene expression in compared with the episomal plasmids. However, the limited availability of suitable loci hinders the extensive metabolic engineering. We here characterized 30 neutral sites in Saccharomyces cerevisiae genome that did not affect cellular fitness by using expression cassettes of green fluorescent protein (eGFP) and fatty acyl-CoA reductase (MaFAR1) with the aid of efficient CRISPR-Cas9 technique. We found that integration of gene expression cassettes to different genome loci resulted a varied GFP signal and fatty alcohol production, which showed that genomic loci could be used for tuning gene expression. The characterized set of neutral sites should be helpful for extensively metabolic engineering of S. cerevisiae for chemical production and other purposes.

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu.

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

Gene-gene interaction of AhRwith and within the Wntcascade affects susceptibility to lung cancer.

BACKGROUND: Aberrant Wnt signalling, regulating cell development and stemness, influences the development of many cancer types. The Aryl hydrocarbon receptor (AhR) mediates tumorigenesis of environmental pollutants. Complex interaction patterns of genes assigned to AhR/Wnt-signalling were recently associated with lung cancer susceptibility. AIM: To assess the association and predictive ability of AhR/Wnt-genes with lung cancer in cases and controls of European descent. METHODS: Odds ratios (OR) were estimated for genomic variants assigned to the Wnt agonist and the antagonistic genes DKK2, DKK3, DKK4, FRZB, SFRP4 and Axin2. Logistic regression models with variable selection were trained, validated and tested to predict lung cancer, at which other previously identified SNPs that have been robustly associated with lung cancer risk could also enter the model. Furthermore, decision trees were created to investigate variant × variant interaction. All analyses were performed for overall lung cancer and for subgroups. RESULTS: No genome-wide significant association of AhR/Wnt-genes with overall lung cancer was observed, but within the subgroups of ever smokers (e.g., maker rs2722278 SFRP4; OR = 1.20; 95% CI 1.13-1.27; p = 5.6 × 10-10) and never smokers (e.g., maker rs1133683 Axin2; OR = 1.27; 95% CI 1.19-1.35; p = 1.0 × 10-12). Although predictability is poor, AhR/Wnt-variants are unexpectedly overrepresented in optimized prediction scores for overall lung cancer and for small cell lung cancer. Remarkably, the score for never-smokers contained solely two AhR/Wnt-variants. The optimal decision tree for never smokers consists of 7 AhR/Wnt-variants and only two lung cancer variants. CONCLUSIONS: The role of variants belonging to Wnt/AhR-pathways in lung cancer susceptibility may be underrated in main-effects association analysis. Complex interaction patterns in individuals of European descent have moderate predictive capacity for lung cancer or subgroups thereof, especially in never smokers.

CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum.

Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.

[Preparation of GH/tPA double transgenic mice and gene expression analysis].

OBJECTIVE: To obtain GH/tPA double transgenic mice, analyze the expression level of tissue plasminogen activator (tPA) in the mammary glands and observe the growth and development of the transgenic mice. METHODS: We obtained the offspring mice of 2 tPA single transgenic mice (P03 and P05) mated with a female nontransgenic mouse by microinjection of linearized GH plasmid into the fertilized eggs and embryo transfer. PCR was used to detect the gene integration. The expression levels of tPA in single gene and double gene transgenic mice were compared using ELISA and Western blotting. We assessed the effects of GH gene transduction on the growth and development of the transgenic mice by observing body weight changes of the mice at each developmental stage. RESULTS: A total of 286 fertilized eggs were collected from P03 mice, and after embryo transfer, 77 offspring mice were obtained, including 16 tPA single transgenic mice (7 male, 9 female) and 13 GH/tPA double transgenic mice (8 male, 5 female) as confirmed by PCR. The integration rate of the double genes was 16.9%. A total of 175 fertilized eggs were collected from P05 mice, and 34 offspring mice were obtained including 12 tPA single transgenic mice (5 male, 7 female) and 7 GH/tPA double transgenic mice (3 male, 4 female), in which the integration rate of the double genes was 20.6%. The highest expression level of tPA in the mammary gland was significantly higher in double than in single transgenic mice (674 µg/mL vs 82.5 µg/mL, P < 0.05). In the whole growth cycle of the mice, no significant difference in weight gain was observed in the single or double transgenic mice as compared with the na?ve mice (P>0.05). CONCLUSION: We successfully prepared GH/tPA double transgenic mice, in which GH gene transduction significantly increases the expression level of target gene tPA without affecting the growth and development of the transgenic mice. This success suggests a promising approach to preparing transgenic animals for producing pharmaceutical proteins and the breeding of the transgenic animals.

Inducible protein expression in piggyBac transposase mediated stable HEK293 cell pools.

Described here is the use of piggyBac transposase generated HEK293 stable cell pools for doxycycline-inducible protein production. The key benefits of the system are that low amounts of plasmid DNA are needed for transfection, high levels of protein expression can be achieved also for toxic proteins at robust scalability and reproducibility and the recombinant cell line can be stored as frozen cell bank. Transfection, selection, expression and purification of enhanced green fluorescence protein (eGFP) and SARS-CoV-2 Spike protein are described in this chapter.

A Novel and Efficient Genome Editing Tool Assisted by CRISPR-Cas12a/Cpf1 for Pichia pastoris.

Pichia pastoris has been widely exploited for the heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value chemicals and pharmaceuticals. Effective synthetic biology tools for genetic engineering are essential for industrial and biotechnological research in this yeast. Here, we describe a novel and efficient genome editing method mediated by the CRISPR-Cpf1 system, which could facilitate the deletion of large DNA fragments and integration of multiplexed gene fragments. The CRISPR-Cpf1 system exhibited a precise and high editing efficiency for single-gene disruption (99 ± 0.8%), duplex genome editing (65 ± 2.5% to 80 ± 3%), and triplex genome editing (30 ± 2.5%). In addition, the deletion of large DNA fragments of 20kb and one-step integration of multiple genes were first achieved using the developed CRISPR-Cpf1 system. Taken together, this study provides an efficient and simple gene editing tool for P. pastoris. The novel multiloci gene integration method mediated by CRISPR-Cpf1 may accelerate the ability to engineer this methylotrophic yeast for metabolic engineering and genome evolution in both biotechnological and biomedical applications.

Multiplex Genome Editing in Yeast by CRISPR/Cas9 - A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways.

Numerous important pharmaceuticals and nutraceuticals originate from plant specialized metabolites, most of which are synthesized via complex biosynthetic pathways. The elucidation of these pathways is critical for the applicable uses of these compounds. Although the rapid progress of the omics technology has revolutionized the identification of candidate genes involved in these pathways, the functional characterization of these genes remains a major bottleneck. Baker's yeast (Saccharomyces cerevisiae) has been used as a microbial platform for characterizing newly discovered metabolic genes in plant specialized metabolism. Using yeast for the investigation of numerous plant enzymes is a streamlined process because of yeast's efficient transformation, limited endogenous specialized metabolism, partially sharing its primary metabolism with plants, and its capability of post-translational modification. Despite these advantages, reconstructing complex plant biosynthetic pathways in yeast can be time intensive. Since its discovery, CRISPR/Cas9 has greatly stimulated metabolic engineering in yeast. Yeast is a popular system for genome editing due to its efficient homology-directed repair mechanism, which allows precise integration of heterologous genes into its genome. One practical use of CRISPR/Cas9 in yeast is multiplex genome editing aimed at reconstructing complex metabolic pathways. This system has the capability of integrating multiple genes of interest in a single transformation, simplifying the reconstruction of complex pathways. As plant specialized metabolites usually have complex multigene biosynthetic pathways, the multiplex CRISPR/Cas9 system in yeast is suited well for functional genomics research in plant specialized metabolism. Here, we review the most advanced methods to achieve efficient multiplex CRISPR/Cas9 editing in yeast. We will also discuss how this powerful tool has been applied to benefit the study of plant specialized metabolism.

Advances on molecular mechanism of hepatitis B virus-induced hepatocellular carcinoma.

The pathogenesis of hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC） is complicated with the crosstalk of multiple factors and the multi-step processes. The main mechanisms underlying the HBV-induced HCC include:①integration of HBV DNA into the host hepatocyte genome to alter gene function at the insertion site，resulting in host genome instability and expression of carcinogenic truncated proteins;②HBV gene mutations at S，C，and X coding regions in the genome;③HBV X gene-encoded HBx protein activates proto-oncogenes and inhibits tumor suppressor genes，leading to the HCC occurrence. In this article，the recent research progress on the molecular mechanism of HBV-induced HCC is comprehensively reviewed，so as to provide insights into the prevention，early prediction and postoperative adjuvant therapy of HCC.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

OBJECTIVE: Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. RESULTS: The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. CONCLUSIONS: A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

Use of Auxotrophic Markers for Targeted Gene Insertions in Trichoderma reesei.

In this chapter, we describe a routinely used strategy for targeted gene insertions in Trichoderma reesei using auxotrophic markers. Generally, targeted gene integrations are advantageous over random, ectopic integration, because the copy number and locus of integration are controlled, abolishing the risk of pleiotropic effects. The use of auxotrophic markers allows a direct, cheap, and easy method for selection. The first step is the construction of recipient strains in a NHEJ-deficient strain. We routinely use deletion strains of pyr4, encoding for the orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) and/or asl1, encoding for the argininosuccinate lyase (EC 4.3.2.1). In the second step, the gene of interest is inserted together with the marker gene. Here we describe the necessary strategy for the construction of the recipient strains and insertion constructs, a PEG-mediated transformation protocol, and a protocol for genetic confirmation of the gene insertion.

Microbial Cell Factory of Baccatin III Preparation in Escherichia coli by Increasing DBAT Thermostability and in vivo Acetyl-CoA Supply.

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbat S189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbat S189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.

Advances on molecular mechanism of hepatitis B virus-induced hepatocellular carcinoma / 浙江大学学报·医学版

The pathogenesis of hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC） is complicated with the crosstalk of multiple factors and the multi-step processes. The main mechanisms underlying the HBV-induced HCC include:①integration of HBV DNA into the host hepatocyte genome to alter gene function at the insertion site，resulting in host genome instability and expression of carcinogenic truncated proteins;②HBV gene mutations at S，C，and X coding regions in the genome;③HBV X gene-encoded HBx protein activates proto-oncogenes and inhibits tumor suppressor genes，leading to the HCC occurrence. In this article，the recent research progress on the molecular mechanism of HBV-induced HCC is comprehensively reviewed，so as to provide insights into the prevention，early prediction and postoperative adjuvant therapy of HCC.