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Variability in genome-engineering source materials: consider your starting point.

Simona Patange1, Sierra D Miller1, Samantha D Maragh1

  • 1Biosystems and Biomaterials Division, Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA.

Synthetic Biology (Oxford, England)
|March 24, 2023
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Summary

Cellular variability impacts genome engineering outcomes. Understanding baseline genotype and phenotype is crucial for reliable and reproducible CRISPR/Cas9 gene editing in mammalian cells.

Keywords:
CRISPR/Cas9genome editinggenome engineeringheterogeneitymetadata

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Area of Science:

  • Cell Biology
  • Genetics
  • Biotechnology

Background:

  • Cellular variability, at both genotype and phenotype levels, is often overlooked in genome engineering.
  • This variability can significantly affect experimental design, execution, and interpretation.

Purpose of the Study:

  • To highlight the impact of cellular variability on genome engineering experiments, particularly using CRISPR/Cas9.
  • To emphasize the importance of characterizing baseline cell properties for accurate interpretation of engineering outcomes.
  • To provide a framework for identifying and mitigating variability in genome engineering.

Main Methods:

  • Perspective piece utilizing clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) genome editing in mammalian cells as a model.
  • Development of a cause-and-effect diagram for CRISPR/Cas9 genome editing to identify variability sources.

Main Results:

  • Variation within and across cell samples can mislead researchers regarding expected outcomes of genome engineering.
  • Characterizing cell heterogeneity and inherent variability is essential for predictable engineered products.
  • A framework is provided to identify and mitigate sources of variability in CRISPR/Cas9 editing.

Conclusions:

  • Researchers must consider source material variability for reliable genome engineering.
  • Strategies to detect, attribute, and minimize variability enhance reproducibility and confidence in genome engineering studies.
  • Accounting for cellular heterogeneity is key to achieving desired and predictable engineered cell products.