Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3-6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures-micronuclei and chromosome bridges-that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

Maintenance of the highly organized striated muscle tissue requires a cell-wide dynamic network that through interactions with all vital cell structures, provides an effective mechanochemical integrator of morphology and function, absolutely necessary for intra- and intercellular coordination of all muscle functions. A good candidate for such a system is the desmin intermediate filament cytoskeletal network. Human desmin mutations and post-translational modifications cause disturbance of this network, thus leading to loss of function of both desmin and its binding partners, as well as potential toxic effects of the formed aggregates. Both loss of normal function and gain of toxic function are linked to mitochondrial defects, cardiomyocyte death, muscle degeneration and development of skeletal myopathy and cardiomyopathy.

The adult myocardium demonstrates a unique system of adaptation upon stress stimuli, in an effort to maintain its overall homeostasis. This compensatory mechanism remains a mystery1. Tumor Necrosis Factor-α (TNF-α) is one of the major stress-induced pro-inflammatory cytokines that is up-regulated in heart failure1,2 and its sustained expression is considered detrimental for the heart1,3–9. Although previous studies have shown that lower levels of TNF-α confer cytoprotection in the myocardium following ischemic reperfusion injury10, such action in heart failure remains elusive. Here we propose a novel cardioprotective function for TNF-α overexpression in a genetic heart failure model, the desmin deficient mice, through NF-κB-mediated cardiomyocyte ectopic expression of keratin 8 (K8) and keratin 18 (K18)11, two simple epithelia-specific Intermediate Filament (IF) proteins. The ectopically expressed K8 and K18 (K8/K18) form a cytoskeletal network that localizes mainly at the Intercalated Discs (IDs). This alternative K8/K18 cytoskeleton confers cardioprotection by a mechanism that maintains ID and mitochondrial integrity and function. Importantly, we demonstrated that K8/K18 ectopic induction takes place in other genetic and experimental models of heart failure and showed a cardioprotective function in mice subjected to transverse aortic constriction. Finally, we discovered that in cardiomyocytes of human failing myocardium, where TNF-α is induced2, K8/K18 are also ectopically expressed and localize primarily at IDs, where desmin cannot be detected. This is the first report to propose a TNFα-mediated cardiac ectopic expression of K8/K18 IF proteins, which may act as stress-induced cardioprotective factors in the failing heart, a phenomenon of major clinical significance as it also extends to human heart failure.

Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.