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- Title
Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations.
- Authors
Grajcarek, Janin; Monlong, Jean; Nishinaka-Arai, Yoko; Nakamura, Michiko; Nagai, Miki; Matsuo, Shiori; Lougheed, David; Sakurai, Hidetoshi; Saito, Megumu K.; Bourque, Guillaume; Woltjen, Knut
- Abstract
The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening. DNA repair by microhomology-mediated end joining creates precise deletions based on flanking microhomologies. Here the authors use CRISPR-Cas9 to recreate pathogenic deletion mutations using existing microhomologies in the human genome identified by their program MHcut.
- Subjects
DNA repair; DOUBLE-strand DNA breaks; HUMAN deletion mutation; CRISPRS; PLURIPOTENT stem cells
- Publication
Nature Communications, 2019, Vol 10, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-019-12829-8