Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations.
Janin GrajcarekJean MonlongYoko Nishinaka-AraiMichiko NakamuraMiki NagaiShiori MatsuoDavid R LougheedHidetoshi SakuraiMegumu K SaitoGuillaume BourqueKnut WoltjenPublished in: Nature communications (2019)
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.
Keyphrases
- dna repair
- induced pluripotent stem cells
- genome wide
- crispr cas
- dna damage
- genome editing
- copy number
- endothelial cells
- dna methylation
- dna damage response
- high glucose
- type diabetes
- molecularly imprinted
- drug induced
- pluripotent stem cells
- adipose tissue
- small molecule
- cancer therapy
- stress induced
- drug delivery
- high resolution