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CRISPR/Cas9 increases mitotic gene conversion in human cells

Gene Therapy volume 27, pages 281–296 (2020)Cite this article

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Abstract

Gene conversion is a process of transferring genetic material from one homologous sequence to another. Most reported gene conversions are meiotic although mitotic gene conversion is also described. When using CRISPR/Cas9 to target the human hemoglobin subunit beta (HBB) gene, hemoglobin subunit delta (HBD) gene footprints were observed in HBB gene. However, it is unclear whether these were the results of gene conversion or PCR-mediated sequence shuffling between highly homologous sequences. Here we provide evidence that the HBD footprints in HBB were indeed results of gene conversion. We demonstrated that the CRISPR/Cas9 facilitated unidirectional sequence transfer from the homologous gene without double-strand breaks (DSB) to the one with DSBs, and showed that the rates of HBD footprint in HBB were positively correlated to the HBB insertion and deletion rates. We further showed that when targeting HBD gene, HBB footprints could also be observed in HBD gene. The mitotic gene conversion was observed not only in immortalized HEK293T cells, but also in human primary cells. Our work reveals mitotic gene conversion as an often overlooked effect of CRISPR/Cas9-mediated genome editing.

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Fig. 1: Experimental strategies.
Fig. 2: Examining sgRNAs for targeting HBB.
Fig. 3: HBD footprints in HBB were the results of gene conversion.
Fig. 4: Gene conversion targeting HBD and human primary cells.

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Data availability

The NGS data generated in this study were submitted to SRA with accession numbers PRJNA575526 and PRJNA590474.

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Funding

This work is supported by the North Carolina State Grant 330054.

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  1. Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA

    Parisa Javidi-Parsijani, Pin Lyu, Vishruti Makani, Walaa Mohamed Sarhan, Kyung Whan Yoo, Lobna El-Korashi, Anthony Atala & Baisong Lu

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Correspondence to Baisong Lu.

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Javidi-Parsijani, P., Lyu, P., Makani, V. et al. CRISPR/Cas9 increases mitotic gene conversion in human cells. Gene Ther 27, 281–296 (2020). https://doi.org/10.1038/s41434-020-0126-z

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