Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR

Ryu, Jae-Yong; Chan, William K.; Wettengel, Jochen M.; Hanna, Carol; Burwitz, Benjamin J.; Hennebold, Jon D.; Bimber, Benjamin N.

doi:10.1016/j.omtm.2022.01.009

Cited by 4 publications

(6 citation statements)

References 33 publications

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“…To further validate the consistency and robustness of the BETLE reporter, we derived clones from BETLE-Pop via single-cell dilution. We performed a previously published modified tagmentation PCR assay to define the number and location of all integrants in the clones and selected both a clone with multiple integrants (BETLE-mult) and a clone with a single integrant (BETLE-sing) (Figure 3a) [28]. This assay confirmed five integration sites in BETLE-mult across multiple chromosomes and a single integration site in BETLE-sing on chromosome 20 (Table S2).…”

Section: Analysis Of Crispr/cas Editing In Betle Reporter Cells With ...mentioning

confidence: 83%

A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing

Wettengel

Hansen-Palmus

Yusova

et al. 2023

IJMS

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CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.

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Section: Analysis Of Crispr/cas Editing In Betle Reporter Cells With ...mentioning

confidence: 83%

A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing

Wettengel

Hansen-Palmus

Yusova

et al. 2023

IJMS

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“…To assess potential insertional mutations for gene therapy, we investigated the integration sites of the MG element in primary T cells. We employed tagmentation-assisted PCR to efficiently recover a large number of mammalian insertion sites, subsequently analyzed via next-generation sequencing ( 63 ). This approach provides a comprehensive and precise approach for investigating transgenic insertion sites and offers the advantages of rapid and straightforward sample preparation, surpassing restriction endonuclease-based techniques.…”

Section: Discussionmentioning

confidence: 99%

“…Genomic DNA from CAR-modified T cells was extracted using the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, China). Enhanced-specificity Tag-PCR was conducted following the published protocol ( 63 ). In brief, tagmentation was performed using 500 ng of gDNA and the Illumina DNA prep kit (Illumina, USA).…”

Section: Methodsmentioning

confidence: 99%

Mage transposon: a novel gene delivery system for mammalian cells

Tian,

Tong,

et al. 2024

Nucleic Acids Research

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Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.

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“…To determine infection efficacy, 10 μL cell culture supernatant was mixed with 100 μL PBS containing 0.1% Tween 20 and 1 μmol/L Coelenterazine N as described. 44 Luminescence was detected using a Tecan Infinite 200 reader (Tecan Group Männedorf, Switzerland).…”

Section: Methodsmentioning

confidence: 99%