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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.

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10-15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of "cancer-selective paralog dependency" may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

Sickle Cell Anemia is a recessive disorder caused by a single point mutation in the human beta globin (HBB) gene. Affecting nearly 1 million people worldwide, this disease is severely lacking in long-term treatment options and is a prime candidate for a gene editing therapeutic approach. Here we report the use of the CRISPR/Cas system to target the human HBB gene in the region of the sickle cell anemia-causing mutation.Utilizing two different Cas9 nickases as well as the wild type nuclease, we are able to introduce blunt double-strand breaks, single strand nicks, and dual-nicks in which the nicks are placed on opposite strands and leave either 3’ or 5’ overhangs of varying lengths. Using either single strand oligonucleotide (ssODN) or plasmid DNA donors, we characterize several different DNA repair outcomes including indel mutations resulting from non-homologous end-joining, homology-dependent repair (HDR) using the donor as a template, and finally HDR using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene. The region of gene conversion includes the sequence most commonly mutated in sickle cell anemia. The frequency of this event depends on the nature of the break. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Sickle Cell Anemia is an inherited recessive disorder caused by a single point mutation in the human beta globin (HBB) gene resulting in an abnormal type of hemoglobin. Here we report targeting the HBB locus using CRISPR/Cas9 technology for correction of Sickle Cell Disease, which affects nearly 1 million people worldwide. Cas9 and its variants can be used to introduce a variety of breaks including blunt double stranded break (DSB), single nicks, or dual nicks leaving either a 3’ or 5’ overhang. The type of cut and donor used can play a role in triggering different repair pathways, thus, resulting in various editing profiles. Using a single strand oligonucleotide (ssODN), we characterize different DNA repair outcomes including indel mutations resulting from Non Homologous End-Joining (NHEJ), Homology-Dependent Repair (HDR) using the donor as a template, and, finally, Gene Conversion (a kind of HDR event) using the closely related HBD gene as an endogenous template. Repair using homologous sequences from the HBD gene results in partial gene-conversion yielding a chimeric HBB-HBD gene that corrects the sickle cell point mutation. We observed that the Cas9 nickase/gRNA pair leaving a 5’ overhang displayed a significantly higher frequency of gene conversion and gene correction than other Cas9-induced DNA end structures. We also provide evidence that overexpression or down-regulation of critical factors in the repair pathways influence the repair pathway balance.In summary, we demonstrate that the frequency of various repair outcomes under different conditions offers insight into the mechanisms of repair of Cas9-induced DNA cleavage. The data support a therapeutic approach in which correction of the sickle-cell mutation is efficiently mediated through HDR using a donor template or by gene-conversion using the endogenous HBD gene.

Epigenetic dysregulation is an emerging hallmark of cancers, and the identification of recurrent somatic mutations in chromatin regulators implies a causal role for altered chromatin states in tumorigenesis. As the majority of epigenetic mutations are inactivating and thus do not present directly druggable targets, we reasoned that these mutations may alter the epigenomic state of cancer cells and thereby expose novel epigenetic vulnerabilities. To systematically search for epigenetic synthetic lethal interactions, we performed a deep coverage pooled shRNA screen across a large collection of cancer cell lines using a library targeting a diverse set of epigenetic regulators. Strikingly, this unbiased screen revealed that silencing of the SWI/SNF ATPase subunit BRM/SMARCA2, selectively inhibits the proliferation of BRG1-deficient cancer cells. The mammalian SWI/SNF complexes (mSWI/SNF) regulate chromatin structure through ATP-dependent nucleosome remodeling. Recent cancer genome studies have revealed a significant frequency of mutations in several components of the mSWI/SNF complexes including loss of the catalytic subunit BRG1 in non-small cell lung cancers. Our studies reveal that BRM knockdown selectively induced cell cycle arrest in BRG1-mutant cancer cells and significantly impaired the growth of BRG1-mutant lung tumor xenografts. BRM is the paralog of BRG1, suggesting a model in which mSWI/SNF mutations lead to a hypomorphic complex that promotes tumorigenesis but cannot tolerate complete inactivation. Therefore, our studies present BRM as an attractive therapeutic target in BRG1-mutant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR06. Citation Format: Zainab Jagani, Gregory Hoffman, Rami Rahal, Frank Buxton, Gregory McAllister, Kay Xiang, Elizabeth Frias, Janina Huber, Alicia Lindeman, Dongshu Chen, Linda Bagdasarian, Rodrigo Romero, Nadire Ramadan, Tanushree Phadke, Kristy Haas, Mariela Jaskelioff, Boris Wilson, Matthew Meyer, Margaret E. McLaughlin, Charles WM Roberts, Vic Myer, Jeff Porter, Nicholas Keen, Craig Mickanin, Frank Stegmeier. A functional screen of the epigenome identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR06.