Short term but highly efficient Cas9 expression mediated by excisional system using adenovirus vector and Cre

Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least 6 months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80–90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.

Short-Term and Highly Efficient Cas9 Expression System Using Adenovirus Vector and Site-Specific Recombinase Cre

Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least six months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80–90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus.

The Expression of Recombinant Human Serum Albumin in the Mammary Gland of Transgenic Mice

Human serum albumin (HSA) is widely used in the clinic for the treatment of several diseases in large amount each year. With the increasing demands of HSA in clinic and limited blood resource, recombinant HSA (rHSA) is becoming an attractive and alternative source for HSA production. In this study, we aimed to express rHSA in the mammary glands of transgenic mice by using a tissue-specific promoter and other regulatory elements. An rHSA expression vector was constructed bearing the cDNA and first intron of HSA under the control of bovine αs1-casein promoter with a 2 × chicken β-globin insulator in the front. Transgenic mice were generated and reverse transcription polymerase chain reaction showed that rHSA was expressed only in the mammary gland, indicating the tissue specificity of the bovine αs1-casein promoter in directing transgene transcription in transgenic mice. Enzyme-linked immunosorbent assay test showed that rHSA was successfully secreted into the milk of transgenic mice with the highest level at 1.98 ± 0.12 g/L. Our results indicate the ability of the bovine αs1-casein promoter to induce successful expression of rHSA in the mammary gland of transgenic mice.

TAEL 2.0: An Improved Optogenetic Expression System for Zebrafish

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. Here, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as “TAEL 2.0.” We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. We believe this improved optogenetic expression system will be a useful resource for the zebrafish community.

Transcription initiation mapping in 31 bovine tissues reveals complex promoter activity, pervasive transcription, and tissue-specific promoter usage

ABSTRACTCharacterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and includes a low number of transcripts in comparison to human and mouse. Using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE data for producing a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-Seq. Lastly, gene co-expression networks revealed an impressive use of tissue-specific promoters, especially between brain and testicle, which expressed several genes in common from alternate transcription start sites. Regions surrounding co-expressed modules were enriched in binding factor motifs representative of their tissues. This annotation will be highly useful for future studies on expression control in cattle and other species. Furthermore, these data provide significant insight into transcriptional activity for a comprehensive set of tissues.

CRISPR/Cas9-Mediated Integration of Large Transgene into Pig CEP112 Locus

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating tool that can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knockout, the gene knock-in (KI) efficiency mediated by homology-directed repair remains low, especially for large fragment integration. In this study, we established an efficient method for the CRISPR/Cas9-mediated integration of large transgene cassette, which carries salivary gland-expressed multiple digestion enzymes (≈ 20 kbp) in CEP112 locus in pig fetal fibroblasts (PFFs). Our results showed that using an optimal homology donor with a short and a long arm yielded the best CRISPR/Cas9-mediated KI efficiency in CEP112 locus, and the targeting efficiency in CEP112 locus was higher than in ROSA26 locus. The CEP112 KI cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that KI pig (705) successfully expressed three microbial enzymes (β-glucanase, xylanase, and phytase) in salivary gland. This finding suggested that the CEP112 locus supports exogenous gene expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus in pigs by using our optimal homology arm system and established a modified pig model for foreign digestion enzyme expression in the saliva.

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

ABSTRACTClustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) is a precise genome manipulating tool which can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knock-out, the gene knock-in efficiency mediated by homology-directed repair (HDR) remains low, especially for large fragment integration. In this study, we established an efficient method for CRISPR/Cas9-mediated integration of large transgene cassette carrying salivary gland-expressing multiple digestion enzymes (≈ 20 kbp) in CEP112 locus in pig fetal fibroblasts. Our results showed that using homologous donor with a short left arm and a long right arm yielded the best CRISPR/Cas9-mediated knock-in efficiency, and the targeting efficiency in potential safe harbor CEP112 locus are higher than ROSA26 locus. The CEP112 knock-in cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that knock-in pig (705) successfully expressed three microbial enzymes (β-glucanase, xylanase, and phytase) in salivary gland, suggesting potential safe harbor CEP112 locus supports exogenous genes expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus using our optimal homology arm system for precise modification of pigs, and established a modified pig model for foreign digestion enzyme expression in saliva.