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RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2′-O-methylation (2′-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2′-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2′-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2′-O-Me can be etiological to human disease. NPM1 regulates ribosomal RNA 2′-O-methylation by binding to small nucleolar RNAs, thereby modulating translation. NPM1 mutations lead to altered 2′-O-methylation and impaired ribosomal function, resulting in bone marrow failure and leukemia susceptibility.

The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies. CRISPR-Cas9 has rapidly become a common molecular biology tool for modifying genomes and has been modified to generate single-strand nicks as well as double-strand breaks. Here the authors explore the DNA repair pathways activated by the different variants of Cas9.

Tumor suppressor p53-binding protein 1 (53BP1) regulates the repair of dysfunctional telomeres lacking the shelterin protein TRF2 by promoting their mobility, their nonhomologous end-joining (NHEJ), and, as we show here, by blocking 5′ resection by CtIP. We report that these functions of 53BP1 required its N-terminal ATM/ATR target sites and its association with H4K20diMe, but not the BRCT domain, the GAR domain, or the binding of 53BP1 to dynein. A mutant lacking the oligomerization domain (53BP1 ᵒˡⁱᵍᵒ) was only modestly impaired in promoting NHEJ of dysfunctional telomeres and showed no defect with regard to the repression of CtIP. This 53BP1 ᵒˡⁱᵍᵒ allele was previously found to be unable to support class switch recombination or to promote radial chromosome formation in PARP1 inhibitor-treated Brca1-deficient cells. The data therefore support two conclusions. First, the requirements for 53BP1 in mediating NHEJ at dysfunctional telomeres and in class switch recombination are not identical. Second, 53BP1-dependent repression of CtIP at double-strand breaks (DSBs) is unlikely to be sufficient for the generation of radial chromosomes in PARP1 inhibitor-treated Brca1-deficient cells.

Background Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations. Results To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290 , and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes. Conclusions UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies. A genome-wide analysis determines the contribution of DNA breaks and nuclear interactions to the formation of random versus recurrent translocations; whereas random translocations follow nuclear interaction profiles, the frequency of recurrent translocations is directly proportional to the amount of DNA damage at translocation partners. DNA breakage and translocation Translocations — events that swap the arms of two different chromosomes — are found in many cancers. It is thought that they occur when the interaction sites become close in nuclear space. Rafael Casellas and colleagues have now done a genome-wide analysis to determine the contribution of DNA breaks to the formation of random and recurrent translocations. Whereas random translocations are found to be highly sensitive to nuclear interactions between chromosomes, the frequency of recurrent translocations, including those involved in human cancer, is proportional to the amount of DNA damage at these highly utilized sites.

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.

The recent correspondence to the Editor of Nature Methods by Schaefer et. al. has garnered significant attention since its publication as a result of its strong conclusions contradicting numerous publications in the field using similar analytical approaches and methods. The authors suggest that the CRISPR-Cas9 system is highly mutagenic in genomic regions not expected to be targeted by the gRNA. Based on experimental design and a re-analysis of the primary data, we believe that the conclusions drawn from this study are unsubstantiated by the disclosed experiments as they were designed and carried out. Further, it is impossible to ascribe the observed differences in the subject mice to the effects of CRISPR per se. The genetic differences seen in this comparative analysis were likely present prior to editing with CRISPR.