Improved and Flexible HDR Editing by Targeting Introns in iPSCs

Fu, Juan; Fu, Ya-Wen; Zhao, Jimin; Yang, Zhouxin; Li, Si-Ang; Li, Guohua; Quan, Zi-Jun; Zhang, Feng; Zhang, Jianping; Zhang, Xiaobing; Sun, Changkai

doi:10.1007/s12015-022-10331-1

Cited by 10 publications

(6 citation statements)

References 43 publications

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“…Since CRISPR/Cas9-mediated KI is a highly efficient and specific targeting method in mammalian cells 20 – 23 , we applied CRISPR/Cas9-mediated KI method to the simHDR system. Previously, a novel MAC vector derived from mouse chromosome 10 (10MAC) was established and showed retention and stability comparable to 11MAC, which was used in both cells and animals as reported 24 .…”

Section: Resultsmentioning

confidence: 99%

Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery

Yamazaki

Matsuo

Okada

et al. 2022

Sci Rep

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Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor containing homology arm by PCR could bypass the cloning steps of target sequence and selection markers. To confirm the functionality of the loaded HDR donors, we constructed a MAC with human leukocyte antigen A (HLA-A) gene in the DT40 cells, and verified the expression of this genomic region by reverse transcription PCR (RT-PCR) and western blotting. Collectively, the simHDR offers a rapid and convenient approach to generate genetically modified models for investigating gene functions, as well as understanding disease mechanisms and therapeutic interventions.

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Section: Resultsmentioning

confidence: 99%

Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery

Yamazaki

Matsuo

Okada

et al. 2022

Sci Rep

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“…Targeting introns in nuclease assisted gene editing approaches has the advantage of minimizing deleterious effects that indels might have at the targeted region. Indeed, editing intronic regions to express a transgene of interest has been reported in multiple studies 23,24,25,26,27 . However, we repurposed this strategy by using a promoter-less transgene that hijacks the regulatory element of the myeloid specific CD11b gene without affecting its endogenous expression.…”

Section: Discussionmentioning

confidence: 99%

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Seclen,

Jang,

Lawal

et al. 2024

Preprint

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Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoter-less intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron ofCD11band observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages.In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUAex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

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“…Moreover, certain introns can greatly enhance gene expression levels. Typically, substances containing introns tend to exhibit much higher expression levels compared to those without introns (Buchman et al 1988, (Clark et al 1993, (Fu et al 2022). In our study, we have identi ed 10 intronic genes (ccmFC, cox2, nad1, nad2, nad4, nad5, nad7, rpl2, rps1, and trnY-GTA) in the C. pubescens mitogenome, totaling to 24 intronic genes.…”

Section: Discussionmentioning

confidence: 99%

The complete mitochondrial genome assembly of Capsicum pubescens reveals key evolutionary characteristics of mitochondrial genes of two Capsicum subspecies

Liu

et al. 2023

Preprint

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Pepper (Capsicum pubescens), one of the five domesticated pepper species, boasts unique characteristics such as numerous hairs on the epidermis of its leaves and stems, black seeds, and vibrant purple flowers. Previously, no studies have reported on the complete assembly of the mitochondrial genome (mitogenome) of C. pubescens. Understanding the mitogenome is crucial for further research on C. pubescens. In our study, we successfully assembled the first mitogenome of C. pubescens, which has been assigned the GenBank accession number OP957066. This mitogenome has a length of 454,165 bp and exhibits the typical circular structure observed in most mitogenomes. We have annotated a total of 70 genes, including 35 protein-coding genes (PCGs), 30 tRNA genes, 3 rRNA genes, and 2 pseudogenes. Compared to the other three pepper mitogenomes (KJ865409, KJ865410, and MN196478), C. pubescens OP957066 exhibits 4 unique PCGs (atp4, atp8, mttB, and rps1), while 2 PCGs (rpl10 and rps3) are absent. It's worth mentioning that each of the three pepper mitogenomes (KJ865409, KJ865410, and MN196478) experienced the loss of 4 PCGs (atp4, atp8, mttB, and rps1). To further explore the evolutionary relationships, we reconstructed a phylogenetic tree using the mitogenomes of C. pubescens and fourteen other species. The structural comparison and synteny analysis of the above four pepper mitogenomes showed that C. pubescens has a higher sequence similarity with KJ865409, and C. pubescens experienced rearrangements with the other three pepper mitogenomes. Interestingly, we observed 72 homologous sequences between the mitochondrial and chloroplast genomes, which accounted for 12.60% of the mitogenome, with a total length of 57,207 bp. These sequences encompassed 12 tRNA genes and the rRNA gene (rrn18). Remarkably, selective pressure analysis suggests that the nad5 gene underwent obvious positive selection. Furthermore, the single base mutation in three genes (nad1, nad2, and nad4) resulted in amino acid change. This study has provided a high-quality mitogenome of pepper, offering valuable molecular data for future investigations into the genetic information exchange between organelle genomes in pepper.

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Improved and Flexible HDR Editing by Targeting Introns in iPSCs

Cited by 10 publications

References 43 publications

Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery

Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

The complete mitochondrial genome assembly of Capsicum pubescens reveals key evolutionary characteristics of mitochondrial genes of two Capsicum subspecies

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