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Updated: Jul 31, 2025

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
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A highly efficient transgene knock-in technology in clinically relevant cell types.

Alexander G Allen1, Samia Q Khan1, Carrie M Margulies1

  • 1Editas Medicine, Cambridge, MA, USA.

Nature Biotechnology
|May 1, 2023
PubMed
Summary

We developed SeLection by Essential-gene Exon Knock-in (SLEEK), a CRISPR-based technology for efficient gene insertion in cell therapies. SLEEK achieves over 90% knock-in efficiency in key cell types, enhancing their therapeutic potential.

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Area of Science:

  • CRISPR gene editing
  • Cellular engineering
  • Gene therapy

Background:

  • Inefficient transgene knock-in hinders the development of advanced cell-based medicines.
  • Current methods often lack the efficiency and specificity required for clinical applications.

Purpose of the Study:

  • To develop a novel CRISPR-based technology for highly efficient and precise transgene knock-in.
  • To enable the integration of therapeutic genes into essential gene loci without compromising cell function.

Main Methods:

  • Utilized a CRISPR nuclease targeting an essential gene exon for precise genomic integration.
  • Designed a cargo template ensuring retained essential gene function post-knock-in.
  • Implemented a negative selection strategy for cells with non-productive insertions/deletions.

Main Results:

  • Achieved over 90% knock-in efficiency in clinically relevant cell types using SeLection by Essential-gene Exon Knock-in (SLEEK).
  • Demonstrated SLEEK's superior efficiency compared to AAV6-mediated TRAC knock-in in T cells, even with non-viral DNA.
  • Engineered natural killer cells with SLEEK knock-in of CD16 and mbIL-15, showing enhanced tumor killing and in vivo persistence.

Conclusions:

  • SLEEK technology significantly improves transgene knock-in efficiency and reliability for cell therapy development.
  • This method maintains cell viability and expansion capacity, crucial for therapeutic applications.
  • SLEEK offers a promising platform for engineering enhanced cell-based therapies, including improved cancer treatments.