Genome editing with engineered nucleases (GEENs) introduce site-specific DNA double-strand breaks (DSBs) and repairs DSBs via nonhomologous end-joining (NHEJ) pathways that eventually create indels (insertions/ deletions) in a genome. Whether the features of indels resulting from gene editing could be customized is asked. A review of the literature reveals how gene editing technologies via NHEJ pathways impact gene editing. The survey consolidates a body of literature that suggests that the type (insertion, deletion, and complex) and the approximate length of indel edits can be somewhat customized with different GEENs and by manipulating the expression of key NHEJ genes. Structural data suggest that binding of GEENsto DNA may interfere with binding of key components of DNA repair complexes, favoring either classical-or alternative-NHEJ. The hypotheses have some limitations, but if validated, will enable scientists to better control indel makeup, holding promise for basic science and clinical applications of gene editing. Also see the video abstract here https://youtu.be/vTkJtUsLi3w
Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.
Gene editing with engineered nucleases introduce double‐strand breaks that are repaired by error‐prone nonhomologous end‐joining (NHEJ). In article number 1900126, Sara G. Trimidal et al. propose that the length and type or resulting indels can now be controlled by editing with different engineered nucleases or by manipulating the expression of NHEJ genes.
Double strand breaks are one of the most lethal forms of DNA lesions that, if left unrepaired can lead to genomic instability, cellular transformation, and cell death. However, cells have two main machineries namely error prone Non homologous end joining repair (NHEJ) or an accurate homology dependent repair to repair the double strand breaks. NHEJ is the preferred mechanism for DNA repair and basically consists of two forms: Canonical (C-NHEJ) and Alternative (A-NHEJ) NHEJ. Our study examined the cellular repair outcome when NHEJ is blocked by targeting two key DNA repair proteins: XRCC4 and MRE-11. We developed an extrachromosomal NHEJ fluorescent reporter assay that uses Transcription activator-like effector nucleases (TALEN) to introduce double strand breaks and detect the NHEJ editing by the presence of GFP fluorescence. We demonstrated the presence of NHEJ editing in the XRCC4(-/-) cells treated with Mirin (a pharmacological inhibitor of MRE-11), albeit with a ~52% efficiency of the normal cells. The transcriptional profiles of the Mirin treated HeLa XRCC4(-/-) cells had 307 uniquely differentially expressed genes that was far greater than HeLa XRCC4(-/-) sample (83 genes) and Mirin treated HeLa cells (30 genes). Pathway analysis unique to the XRCC4(-/-) +Mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that the double strand DNA repair can be sustained even in absence of key DNA repair proteins XRCC4 and MRE-11.
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