An unbiased genome-wide method reveals on- and off-target DNA cleavage by TALEN and Cas9 nucleases by detecting chromosome translocation events. Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G ₁ phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna ^-/- cells resemble Rb ^-/- cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.

Mutations within LMNA, encoding A-type nuclear lamins, are associated with multiple tissue-specific diseases, including Emery-Dreifuss (EDMD2/3) and Limb-Girdle muscular dystrophy (LGMD1B). X-linked EDMD results from mutations in emerin, a lamin A-associated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) down-regulated for either A-type lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking A-type lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and M-cadherin; up-regulated Myf5; but no changes in Pax3, Pax7, MEF2C, MEF2D, c-met, and β-catenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna^–/– myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna^–/– myoblasts resulted in increased differentiation potential. These studies indicate roles for A-type lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/C-deficient and emerin-deficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (HBB). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)–mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34⁺ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.