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Gene conversion following CRISPR/Cas9 DNA cleavage: an overlooked effect

Gene Therapy volume 27, pages 245–246 (2020)Cite this article

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In the last few years the rapid pace in the development of gene editing technology has sparked hopes for curing rare diseases, leading to a potentially transformative impact in the field of gene therapy. Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) gene editing technology consists of a bacterial endonuclease (Cas9) complexed with a small guide RNA matching a 20 nucleotide target DNA sequence in the genome, with this complex specifically recognizing the target site with a protospacer adjacent motif (PAM) sequence [1]. The cleavage of this target-genomic sequence generates a double-stranded break of the DNA and initiation of the cellular repair machinery to use, or not use, a repair template. The DNA repair outcome is respectively a non-precise editing (indels) or precise editing via non homologous end-joining or homology-directed repair (HDR) [2]. To date according to ClinicalTrial.gov database from the National Institute of Health, over 20 phase I clinical trials are currently underway and some have promising outcomes. Ensuring the safety of the CRISPR/Cas9 gene editing approach is therefore paramount to its implementation for ex-vivo or in-vivo editing in the clinics. This translates to ensuring high specificity and efficiency in targeting.

While the focus of therapeutic gene editing technology has been heavily focused on off-target effects and the control of Cas9 delivery to ensure safety and efficacy in targeting, an overlooked and important aspect of CRISPR editing is the occurrence of mitotic gene conversion at the target site. Gene conversion consists of, following homologous recombination, the transfer of DNA from one genomic location without double-stranded break of the DNA, to another one location where the DNA has been cleaved. The importance of gene conversion and, in general, the use of an endogenous repair template for gene editing experiments came to light recently following the highly publicized Ma et al. [3] observation on human embryos. This set of experiments consisted of repairing a dominant mutation in MYBPC3 gene, responsible for a severe congenital hypertrophic cardiopathy. The authors repaired the mutation in the father’s genome using HDR repair with delivery of Cas9 ribonucleoprotein and a repair template to the fertilized embryos. They reported a high proportion of repaired alleles indicating the success of the approach. Surprisingly, the authors noted some edited embryos had the maternal allele present on the paternal target strand, suggesting the maternal allele was used as an HDR repair template. A hypothesis formulated by the authors is that the maternal allele was exchanged through inter-homology repair. While this interpretation is still highly disputed [4, 5], this leads to a series of intriguing research questions. How, or why, does the repair machinery choose to use an endogenous repair template following Cas9 cleavage and how prevalent is the use of an endogenous repair template foe gene conversion? If gene conversion is more prevalent than originally thought, would it be possible to use homologous regions in the genome as repair templates?

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Funding

GB receives funding from the National collaborative Research Infrastructure (NCRIS) via the Australian Phenomics Network, National Health and Medical Research Council of Australia (APP1143008), the Australian Research Council (DP180101494), and the National Natural Science Foundation of China (81772214).

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  1. Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia

    Gaetan Burgio

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Burgio, G. Gene conversion following CRISPR/Cas9 DNA cleavage: an overlooked effect. Gene Ther 27, 245–246 (2020). https://doi.org/10.1038/s41434-020-0154-8

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