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Efficient multitool/multiplex gene engineering with TALE-BE.

Alex Boyne1, Ming Yang1, Sylvain Pulicani2

  • 1Cellectis Inc., New York, NY, United States.

Frontiers in Bioengineering and Biotechnology
|November 28, 2022
PubMed
Summary
This summary is machine-generated.

Transcription activator-like effector (TALE) base editors directly convert DNA cytosine to thymine. This study reveals TALE base editor rules in cells, achieving high CD52 gene knockout and enabling multiplex genome engineering with minimal byproducts.

Keywords:
TALEbase editorscell engineeringgene editingt-cells

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Area of Science:

  • Molecular Biology
  • Genome Engineering
  • Biochemistry

Background:

  • TALE base editors (TALE-BE) are novel tools for direct DNA editing, converting cytosine (C) to thymine (T).
  • Understanding the precise editing rules of TALE-BE is crucial for their effective application in genome engineering.

Purpose of the Study:

  • To dissect the editing rules of TALE base editors (TALE-BE) within a cellular context.
  • To optimize TALE-BE for efficient gene targeting and multiplex genome engineering.

Main Methods:

  • Screening of numerous TALE-BE targeting nuclear genomic loci.
  • High-throughput strategy employing precise knock-in of TALE-BE target site collections into the cell genome for in cellulo analysis.
  • Design and application of TALE-BE for CD52 gene knockout and combination with TALEN for multiplex editing.

Main Results:

  • In-depth insights into TALE-BE editing rules were obtained, excluding confounding genomic locus variations.
  • Achieved high-frequency CD52 gene knockout (up to 80% phenotypic knockout) using optimized TALE-BE.
  • Demonstrated minimal generation of indels and byproducts by TALE-BE.
  • Successfully performed multiplex genome engineering with high double gene knockout efficiency (approx. 75%) without translocations.

Conclusions:

  • TALE base editors can be effectively utilized to understand and apply precise DNA editing rules in cellular systems.
  • TALE-BE technology enables efficient gene knockout and advanced multiplex genome engineering with high accuracy and minimal off-target effects.