Carissa Klanseck

Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. Here, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process we refer to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.

Significance: When sound vibrations reach the inner ear, fine protein filaments called “tip links” stretch and open cochlear hair-cell mechanosensitive channels that trigger sensory perception. Similarly, vestibular hair cells use tip links to sense mechanical stimuli produced by head motions. Tip links are formed by cadherin-23 and protocadherin-15, two large proteins involved in hearing loss and balance disorders. Here we present multiple structures, models, and simulations that depict the lower end of the tip link, including the complete protocadherin-15 ectodomain. These models show an essential connection between cadherin-23 and protocadherin-15 with dual molecular “handshakes” and various protein sites that are mutated in inherited deafness. The simulations also reveal how the tip link responds to force to mediate hearing and balance sensing.