Pseudogene-Mediated Gene Conversion After CRISPR-Cas9 Editing Demonstrated by Partial CD33 Conversion with SIGLEC22P

Area of Science:

• Molecular Biology
• Genetics
• Immunology

Background:

• CRISPR-Cas9 gene editing aims for precise DNA modification.
• Off-target edits are a known concern, but pseudogene interference is not well-documented.

Purpose of the Study:

• To investigate the CRISPR-Cas9 strategy for targeted exon 2 excision in the CD33 gene.
• To identify and characterize unexpected outcomes in gene editing, specifically concerning pseudogene involvement.

Main Methods:

• Utilized CRISPR-Cas9 for targeted deletion of exon 2 in the CD33 gene within U937 human monocyte cell line.
• Screened candidate cell lines using the P67.6 antibody (gemtuzumab) which targets the CD33 IgV domain.
• Performed sequencing to analyze genetic alterations in unexpected P67.6-negative cell lines.

Main Results:

• Observed unexpected P67.6-negative cell lines that retained CD33 exon 2.
• Sequencing revealed gene conversion from the SIGLEC22P pseudogene during homology repair, introducing three missense mutations.
• Ectopic expression studies confirmed these mutations disrupt the P67.6 epitope.

Conclusions:

• Pseudogene-directed homology repair represents a novel mechanism for aberrant CRISPR-Cas9 gene editing outcomes.
• This finding highlights a previously unrecognized challenge in gene editing specificity and accuracy.
• The study underscores the importance of considering pseudogene interactions in designing precise gene editing strategies.