Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 enables precise genome editing.
• Standard experiments involve cell editing followed by cloning.
• Cloned cells are presumed to be isogenic.

Purpose of the Study:

• To analyze off-target edits from CRISPR-Cas9.
• To quantify culture-acquired mutations and CNAs in cloned cell lines.
• To assess genomic divergence in edited vs. unedited clones.

Main Methods:

• Whole genome sequencing of three independent CRISPR-Cas9 experiments.
• Analysis of genomic loci, off-target edits, single nucleotide mutations, and copy number alterations (CNAs).
• Comparison of genomic profiles across multiple clones from each experiment.

Main Results:

• Off-target CRISPR-Cas9 edits were minimal.
• Hundreds to thousands of unique single nucleotide mutations were detected per clone after 10-20 passages.
• Copy number alterations (CNAs) of kb to mb size were the primary source of genomic divergence among clones.

Conclusions:

• Screening clones for culture-acquired mutations and CNAs is crucial for accurate interpretation of genome editing experiments.
• Given inevitable culture-associated mutations, experiments should compare mixed unedited and mixed edited clonal lines.