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

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.