The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.

Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors

Targeting the DNA Damage Response in Cancer

The major pathways of DNA double-strand break (DSB) repair are crucial for maintaining genomic stability. However, if deployed in an inappropriate cellular context, these same repair functions can mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. The two major mechanisms of DSB repair in mammalian cells are non-homologous end joining (NHEJ) and homologous recombination. In this Review, we consider DSB repair-pathway choice in somatic mammalian cells as a series of 'decision trees', and explore how defective pathway choice can lead to genomic instability. Stalled, collapsed or broken DNA replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing repair-pathway choice at stalled replication forks differ from those at replication-independent DSBs.

DNA double-strand break repair-pathway choice in somatic mammalian cells.

Approximately 1-5% of breast cancers are attributed to inherited mutations in BRCA1 or BRCA2 and are selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. In other cancer types, germline and/or somatic mutations in BRCA1 and/or BRCA2 (BRCA1/BRCA2) also confer selective sensitivity to PARP inhibitors. Thus, assays to detect BRCA1/BRCA2-deficient tumors have been sought. Recently, somatic substitution, insertion/deletion and rearrangement patterns, or 'mutational signatures', were associated with BRCA1/BRCA2 dysfunction. Herein we used a lasso logistic regression model to identify six distinguishing mutational signatures predictive of BRCA1/BRCA2 deficiency. A weighted model called HRDetect was developed to accurately detect BRCA1/BRCA2-deficient samples. HRDetect identifies BRCA1/BRCA2-deficient tumors with 98.7% sensitivity (area under the curve (AUC) = 0.98). Application of this model in a cohort of 560 individuals with breast cancer, of whom 22 were known to carry a germline BRCA1 or BRCA2 mutation, allowed us to identify an additional 22 tumors with somatic loss of BRCA1 or BRCA2 and 47 tumors with functional BRCA1/BRCA2 deficiency where no mutation was detected. We validated HRDetect on independent cohorts of breast, ovarian and pancreatic cancers and demonstrated its efficacy in alternative sequencing strategies. Integrating all of the classes of mutational signatures thus reveals a larger proportion of individuals with breast cancer harboring BRCA1/BRCA2 deficiency (up to 22%) than hitherto appreciated (∼1-5%) who could have selective therapeutic sensitivity to PARP inhibition.

/pdf/hrdetect-is-a-predictor-of-brca1-and-brca2-deficiency-based-3xgfww3hoc.pdf

HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures

Proteins of the Fanconi anaemia pathway are master regulators of genomic integrity through their interactions with other DNA repair pathways to repair interstrand crosslinks, stabilize replication forks and regulate cytokinesis.

The Fanconi anaemia pathway: new players and new functions

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions.

/pdf/homologous-recombination-and-human-health-the-roles-of-brca1-10ub5n58cv.pdf

Homologous Recombination and Human Health: The Roles of BRCA1, BRCA2, and Associated Proteins

Germ-line and somatic mutations in genes that promote homology-directed repair (HDR), especially BRCA1 and BRCA2, are frequently observed in several cancers, in particular, breast and ovary but also prostate and other cancers. HDR is critical for the error-free repair of DNA double-strand breaks and other lesions, and HDR factors also protect stalled replication forks. As a result, loss of BRCA1 or BRCA2 poses significant risks to genome integrity, leading not only to cancer predisposition but also to sensitivity to DNA-damaging agents, affecting therapeutic approaches. Here we review recent advances in our understanding of BRCA1 and BRCA2, including how they genetically interact with other repair factors, how they protect stalled replication forks, how they affect the response to aldehydes, and how loss of their functions links to mutation signatures. Importantly, given the recent advances with poly(ADP-ribose) polymerase inhibitors (PARPi) for the treatment of HDR-deficient tumors, we discuss mechanisms by which BRCA-deficient tumors acquire resistance to PARPi and other agents.

Homology-Directed Repair and the Role of BRCA1, BRCA2, and Related Proteins in Genome Integrity and Cancer.

Mutations in the tumor suppressor BRCA2 predominantly predispose to breast cancer. Paradoxically, while loss of BRCA2 promotes tumor formation, it also causes cell lethality, although how lethality is triggered is unclear. Here, we generate BRCA2 conditional non-transformed human mammary epithelial cell lines using CRISPR-Cas9. Cells are inviable upon BRCA2 loss, which leads to replication stress associated with under replication, causing mitotic abnormalities, 53BP1 nuclear body formation in the ensuing G1 phase, and G1 arrest. Unexpected from other systems, the role of BRCA2 in homologous recombination, but not in stalled replication fork protection, is primarily associated with supporting human mammary epithelial cell viability, and, moreover, preventing replication stress, a hallmark of pre-cancerous lesions. Thus, we uncover a DNA under replication-53BP1 nuclear body formation-G1 arrest axis as an unanticipated outcome of homologous recombination deficiency, which triggers cell lethality and, we propose, serves as a barrier that must be overcome for tumor formation. BRCA2 mutations promote tumour formation while also paradoxically causing cell lethality. Here the authors generate conditional BRCA2 loss in a non-transformed human mammary cell line and see increased replication stress due to under-replication of DNA.

/pdf/brca2-suppresses-replication-stress-induced-mitotic-and-g1-1ahilxqkv9.pdf

BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

BRCA2 is a breast and ovarian tumor suppressor that guards against genome instability, a hallmark of cancer. Significant progress has been made in improving our understanding of BRCA2 function from biochemical, cellular, and mouse studies. The knowledge gained has been actively exploited to develop therapeutic strategies, including PARP inhibition, which has shown promising clinical outcomes. Recently, tremendous excitement has been generated by the findings of the roles of BRCA2 and other proteins in suppressing replication stress through homologous recombination and in the protection of stalled replication forks. Processes such as mitotic DNA synthesis and fork reversal have taken center stage in these studies. Here, we discuss our recent findings in the context of these advances.

/pdf/homologous-recombination-and-replication-fork-protection-3a7oely81r.pdf

Homologous Recombination and Replication Fork Protection: BRCA2 and More!

Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.

https://www.pnas.org/content/pnas/114/14/3696.full.pdf

Weiran Feng

Papers

Homologous Recombination and Human Health: The Roles of BRCA1, BRCA2, and Associated Proteins

Homology-Directed Repair and the Role of BRCA1, BRCA2, and Related Proteins in Genome Integrity and Cancer.

BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination

Homologous Recombination and Replication Fork Protection: BRCA2 and More!

CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.