Homology-Directed Repair (HDR) Knock-in Templates

Resources

Non-viral delivery promises a true alternative for cell therapy developers

Feature article on benefits and methods for overcoming challenges

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• GenExact™ ssDNA: High efficient and low cytotoxicity non-viral T cell engineering process at clinical scale

  Collaboration with Marson's lab from UCSF (University of California San Francisco) showed that:

  • GenExact™ ssDNA consistently outperformed in-house generated HDRTs (homology-directed repair templates), showing lower levels of toxicity and higher knock-in efficiencies;
  • The KI efficiency of GenExact™ ssDNA via electroporation without any enhancer can reach to 46.2% at a GMP-compatible scale;
  • KI positive live cells count generated by GenExact™ ssDNA was way above estimated patient dose (100*10e6). In vitro assays demonstrated efficient killing of BCMA-CAR cells.

  

  Brian R Shy. et.al., bioRxiv(2021), Read the whole article.

• GenWand™ dsDNA: Outperforms PCR product in knocking in larger genes

  GenWand™ dsDNA demonstrated 80% higher KI efficiency compare to in-house PCR dsDNA (38% vs 21%)

  

  GFP KI at RAB11A Locus in HEK293 cells using electroporation with 5ug of PCR dsDNA and GenWand™ dsDNA templates.

• GenCircle™ dsDNA: Ideal replacement of plasmid HDR template with higher KI efficiency

  GenCircle dsDNA:

  • KI efficiency increased by up to  30% compared to standard plasmid
  • High transfection and low cytotoxicity due to only 429bp vector backbone
  • Avoid drug resistance and regulatory concern as both the antibiotic resistance gene and bacterial origin sequence have been removed

  • CRISPR KI template（ 2.5 kb insertion）
  

• Case studies

  Long hybrid ssDNA HDR templates enable high yield non-viral cell therapy manufacturing

  High Gene knock-in Efficiency and Reduced off Target Integration with single-stranded DNA HDR Template

  More CRISPR Case Studies and FAQs

• KI protocols

  CRISPR Knock-in Protocol for HEK 293T/293 cells with Thermo Fisher Neon® Transfection System

  CRISPR Knock-in Protocol for Jurkat cells with Thermo Fisher Neon® Transfection System

  CRISPR Knock-in Protocol for stimulated Human T Cells with Lonza 4D Nucleofector™ X Unit

  CRISPR Knock-in Protocol for stimulated Human T Cells with Maxcyte® Electroporation System

  Design and Preparation Guidelines for CRISPR KI HDR Templates

• HDR flyer
  

  HDR flyer

  Non-viral HDR Templates Flyer

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