Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system

Ma, Ming; Zhuang, Fang‐Dong; Hu, Xiongbing; Wang, Bolun; Xi, Wen; Ji, Jiafu; Xi, Jianzhong

doi:10.1038/cr.2017.29

Cited by 93 publications

(78 citation statements)

References 15 publications

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“…Ma et al. () reported improvements in precise repair using an avidin‐Cas9 fusion protein with biotinylated repair templates. Improvements in reporter allele generation have also been reported using this approach (Gu, Posfai, & Rossant, ).…”

Section: Commentarymentioning

confidence: 93%

Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

Gertsenstein

Nutter

2018

CP Mouse Biology

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Mice carrying patient-associated point mutations are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence-the orthologous sequence for point mutations or the N or C terminus for epitope tags-appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening of born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target mutations introduced by Cas9. © 2018 by John Wiley & Sons, Inc.

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Section: Commentarymentioning

confidence: 93%

Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

Gertsenstein

Nutter

2018

CP Mouse Biology

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“…The notion that homology search is limited by the distance between the DSB site and the recombination donor was at the basis of the tethering of the donor DNA to Cas9 and more recently of the targeting of the donor to Cas9 breaks by the Fkh1 protein from S. cerevisiae, two approaches that significantly increased genome-editing efficiency (Ma et al 2017;Gu et al 2017;Savic et al 2017;Roy et al 2018). In some cases, modulating mobility and/ or resection efficiency may also be key to successful Cas9 editing (Charpentier et al 2018).…”

Section: Resultsmentioning

confidence: 99%

Keep moving and stay in a good shape to find your homologous recombination partner

Bordelet

Dubrana

2018

Curr Genet

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Genomic DNA is constantly exposed to damage. Among the lesion in DNA, double-strand breaks (DSB), because they disrupt the two strands of the DNA double helix, are the more dangerous. DSB are repaired through two evolutionary conserved mechanisms: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Whereas NHEJ simply reseals the double helix with no or minimal processing, HR necessitates the formation of a 3′ssDNA through the processing of DSB ends by the resection machinery and relies on the recognition and pairing of this 3′ssDNA tails with an intact homologous sequence. Despite years of active research on HR, the manner by which the two homologous sequences find each other in the crowded nucleus, and how this modulates HR efficiency, only recently emerges. Here, we review recent advances in our understanding of the factors limiting the search of a homologous sequence during HR.

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“…As this is a limiting factor in the overall efficacy of CAR T cell therapies, bioengineering strategies to improve gene transfer are in high demand. New nanomaterials based on biotin‐streptavidin conjugation have been used to deliver and link donor templates to Cas9 in human cells, improving rates of gene transfer by 5‐fold relative to conventional methods . Other labs have directly modified Cas9 protein to achieve high‐fidelity edits without off‐target effects, as well as recognize a wider range of potential editing sites, thus improving both the safety and versatility of the system .…”

Section: Car Gene Transfer and Editingmentioning

confidence: 99%

Bioengineering Solutions for Manufacturing Challenges in CAR T Cells

Piscopo

Mueller

Das

et al. 2017

Biotechnology Journal

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The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed “living drugs” for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.

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Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system

Cited by 93 publications

References 15 publications

Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

Keep moving and stay in a good shape to find your homologous recombination partner

Bioengineering Solutions for Manufacturing Challenges in CAR T Cells

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