Kidney ECM Pregel Nanoarchitectonics for Microarrays to Accelerate Harvesting Gene-Edited Porcine Primary Monoclonal Spheres

Gao, Mengyu; Zhu, Xinglong; Peng, Wanliu; He, Yuting; Li, Yang; Wu, Qiong; Zhou, Yanyan; Liao, Guangneng; Yang, Guang; Bao, Ji; Bu, Hong

doi:10.1021/acsomega.2c01074

Cited by 7 publications

(9 citation statements)

References 63 publications

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“…To efficiently apply the CRISPR/Cas9 mRNA form to editing endogenous genes in pigs, we further utilized it to the knockout of the acute rejection gene β4GalNT2 , and harvested the gene‐edited monoclonal cells with β4GalNT2 knockout through DKEM micropattern arrays established by our previously reported study (Figure 4A). [ 16 ] Four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form. DKEM micropattern arrays are tissue‐specific and facilitate efficient culture and harvesting.…”

Section: Resultsmentioning

confidence: 99%

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells

Peng,

Gao,

Zhu

et al. 2024

Biotechnology Journal

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CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene‐edited pigs. The inefficiency in acquiring gene‐edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene‐edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

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Section: Resultsmentioning

confidence: 99%

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells

Peng,

Gao,

Zhu

et al. 2024

Biotechnology Journal

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“…Porcine primary kidney fibroblasts were isolated from the newborn Bama piglets as the nuclear donor as described in the previous method (Gao et al, 2022). The perivitelline space of enucleated oocytes was then injected with a solitary fibroblast donor cell.…”

Section: Somatic Cell Nuclear Transfer and Embryo Culturementioning

confidence: 99%

“…Subsequently, we identified and selected three specific sgRNA sequences, which were cloned into the PX458 vector. These constructs were then transfected into porcine kidney fibroblasts (Gao et al, 2022), and cultured with 10% fetal bovine serum (Royacel, China). Genomic DNA was extracted from the cells 7 days post-transfection (Qiagen, Germany), and the target sequences were amplified and forwarded to PCR for Sanger sequencing using specific primers (Fw 5′-3′CACAATGGTTTG TCCCTG; Rv 3′-5′ ATCATTTCCGTTCCTACT).…”

Section: Sgrna Screening For Target Genementioning

confidence: 99%

One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses

Gao,

He,

Zhu

et al. 2024

Front. Cell Dev. Biol.

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Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV).Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated.Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9.Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

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“…Single clones of miR-17-92 locus cells were picked by microarray [15], and after they grew, genomic DNA was extracted from them. PCR was performed with primer M1, followed by agarose gel electrophoresis analysis to confirm successful integration.…”

Section: Targeted Integration Of Shrna Genesmentioning

confidence: 99%

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV

Mao,

Li,

Gao

et al. 2024

Preprint

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In the world's first pig-to-human cardiac xenotransplant, the elevated levels of porcine cytomeg-alovirus (PCMV) are considered key factors contributing to patient mortality. This has renewed attention to PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effec-tively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infec-tion capabilities. This opens up possibilities for the future production of pig populations with anti-viral functionalities.

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Kidney ECM Pregel Nanoarchitectonics for Microarrays to Accelerate Harvesting Gene-Edited Porcine Primary Monoclonal Spheres

Cited by 7 publications

References 63 publications

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells

One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV

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