CRISPR/Cas9 increases mitotic gene conversion in human cells.

Area of Science:

• Molecular Biology
• Genetics
• Genome Editing

Background:

• Gene conversion is a DNA repair process transferring genetic information between homologous sequences.
• While meiotic gene conversion is well-documented, mitotic gene conversion is less understood.
• CRISPR/Cas9 targeting of the human beta-globin gene (HBB) revealed unexpected hemoglobin delta-globin gene (HBD) sequences.

Purpose of the Study:

• To investigate whether observed HBD footprints in the HBB gene during CRISPR/Cas9 editing result from gene conversion or PCR artifacts.
• To elucidate the mechanism and frequency of mitotic gene conversion induced by CRISPR/Cas9 genome editing.

Main Methods:

• CRISPR/Cas9 was used to target the HBB and HBD genes in human cell lines (HEK293T and primary cells).
• Analysis of sequence data to identify HBD footprints in HBB and vice versa.
• Correlation analysis between footprint rates and insertion/deletion rates at target sites.
• Assessment of gene conversion in both immortalized and primary human cells.

Main Results:

• Evidence confirmed that HBD footprints in HBB were due to gene conversion, not PCR-mediated shuffling.
• CRISPR/Cas9 facilitated unidirectional gene conversion from a non-double-strand break (DSB) site to a DSB site.
• The rate of HBD footprints in HBB correlated positively with HBB insertion and deletion rates.
• Mitotic gene conversion was observed in both HEK293T and human primary cells when targeting HBD or HBB.

Conclusions:

• Mitotic gene conversion is a significant, often overlooked, consequence of CRISPR/Cas9 genome editing.
• CRISPR/Cas9 can induce gene conversion even without a double-strand break at the donor locus.
• This finding has implications for genome editing safety and accuracy, particularly in therapeutic applications.