![]() ![]() While wild-type Cas9 has been proven a powerful tool to profile DSB repair outcomes like endpoint mutations, chemically and optically inducible Cas9 systems provide the fine temporal control needed to quantitatively study the dynamics of early repair events within the first minutes of repair. Finally, we Understanding DSB repair dynamics using inducible Cas9 systems This section highlights new biological insights gained by the implementation of Cas9 systems to study templated and non-templated repair, followed by a discussion on the utility of high-throughput Cas9 screens to characterize DSB repair networks. The ease of reprogramming Cas9 against genomic targets has empowered researchers to precisely probe the molecular mechanisms of DSB repair for various DNA targets, flanking chromatin sequences/modifications, and genetic backgrounds. Doxycycline was one of the first chemically inducible systems used to transcriptionally induce CRISPR-Cas9 expression and was successfully used in early CRISPR screen Understanding the biology of DNA DSB repair using Cas9 systems Current inducible systems mainly include chemically and optically inducible CRISPR-Cas9s, although local genome editing has also been demonstrated using magnetic particles. Inducible Cas9s allow spatiotemporal control of Cas9 cleavage activity by either transcriptional control or post-transcriptional activation. Advances in inducible CRISPR-Cas9 systems For example, chemical mutagens are simple to implement and induce DSBs with high efficiency but produce damage at genetically undefined locations, making it impossible to study DDR on predetermined genomic targets without causing excessive damage to the cell. ![]() The use of physical irradiation, chemical mutagens, and chimeric nucleases as exogenous DSB inducers has contributed significantly to our knowledge of DSB repair, and each technique has strengths and weaknesses (details provided in Box 2). To prevent the accumulation of DNA damage and mutations, cells have evolved a dynamic network of proteins to detect, signal, and repair various genomic lesions, CRISPR-Cas9 as a programmable DSB inducer Failure to quickly repair DNA lesions can lead to permanent genomic mutation and disrupted cellular function, ultimately causing physiological dysregulation and disease. In human cells, genomic DNA spontaneously accumulates lesions as a result of replication stress, endogenous metabolites, and environmental carcinogens. ![]() Section snippets DNA double-strand break repair When combined with sequencing and genome-specific imaging approaches, inducible Cas9 systems greatly expand our capability to spatiotemporally characterize cellular responses to DSB at specific genomic coordinates, providing mechanistic insights that were previously unobtainable. We highlight rapidly inducible Cas9 systems that enable synchronized and efficient break induction. ![]() This review surveys the latest methodological advances and biological insights gained by utilizing Cas9 as a precise ‘damage inducer’ for the study of DSB repair. Pioneering studies have identified and characterized many crucial repair factors in this network, while the advent of genome manipulation tools like clustered regularly interspersed short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) has reinvigorated interest in DSB repair mechanisms. To combat genomic instability caused by DSBs, evolution has outfitted cells with an intricate protein network dedicated to the rapid and accurate repair of these lesions. DNA double-strand breaks (DSBs) are one of the most genotoxic DNA lesions, driving a range of pathological defects from cancers to immunodeficiencies. ![]()
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