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.

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.

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.

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.

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.

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).
CONCLUSIONS: 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.

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.

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.

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.

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.