The CRISPR-Cas9 system has been successfully applied in many organisms as a powerful genome-editing tool. Undoubtedly, it will soon be applied to human genome editing, including gene therapy. Researchers from the National Institute of Health Sciences, Japan have previously reported that unintentional DNA sequences derived from retrotransposons, genomic DNA, mRNA and vectors are captured at double-strand breaks (DSBs) sites when DSBs are introduced by the CRISPR-Cas9 system. Therefore, it is possible that unintentional insertions associated with DSB repair represent a potential risk for human genome editing gene therapies. To address this possibility, comprehensive sequencing of DSB sites was performed. Here, the researchers report that exosome-mediated horizontal gene transfer occurs in DSB repair during genome editing. Exosomes are present in all fluids from living animals, including seawater and breathing mammals, suggesting that exosome-mediated horizontal gene transfer is the driving force behind mammalian genome evolution. The findings of this study highlight an emerging new risk for this leading-edge technology.
Trans-species horizontal gene transfer at the Peg10 gene locus
from the serum included in the culture medium
a Distribution of indels at CRISPR-Cas9-induced DSB sites in NIH-3T3 cells cultured using DMEM containing 10% goat serum instead of FBS. In addition, 38% of the sequence reads were deletions. Large insertions (greater than 33 bp) represented 4% of the total sequence reads (red region). b Here, 51% of the large insertions corresponded to partial sequences of the plasmid DNA that was transfected into the NIH-3T3 cells. In addition, 16% and 1% of the reads were identical to mouse genomic DNA and mRNA sequences (MM10), respectively. Moreover, 29% of the large insertions corresponded to E. coli genomic DNA. The remaining 3% of the total reads are described in c (blue region). c Approximately 9% of the reads classified as others were from goat, including the goat genome and goat SINEs and goat satellite DNA. Structures of de novo inserted goat sequences at the Peg10-ORF1 loci (d, e). Both the postintegration site and preintegration sequences (bottom of the panel) are presented. The nucleotide sequences that correspond to the single guide RNA sequence and the PAM sequences are presented in red and bold red characters, respectively. The black lines indicate the junction sites between pre- and postintegration sequences. The sequences in the blue boxes are overlapping microhomologies and are marked with black dotted lines. Each insertion was truncated at both the 5′ and 3′ ends. d Partial goat DNA sequences from chromosome 28 were inserted with a 1-bp microhomology. e A truncated goat satellite DNA sequence was inserted with a 2-bp overlapping microhomology
Ono R, Yasuhiko Y, Aisaki KI, Kitajima S, Kanno J, Hirabayashi Y. (2019) Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing. Commun Biol 2:57. [article]