DNA repair in cultured mouse cells of increasing population doubling level

DNA ligase I is the key ligase for DNA replication in mammalian cells and has also been reported to be involved in a number of recombination and repair processes. Our previous finding that Lig1 knockout mouse embryos developed normally to mid-term before succumbing to a specific haematopoietic defect was difficult to reconcile with a report that DNA ligase I is essential for the viability of cultured mammalian cells. To address this issue, we generated a second Lig1 targeted allele and found that the phenotypes of our two Lig1 mutant mouse lines are identical. Widely different levels of Lig1 fusion transcripts were detected from the two targeted alleles, but we could not detect any DNA ligase I protein, and we believe both are effective Lig1 null alleles. Using foetal liver cells to repopulate the haematopoietic system of lethally irradiated adult mice, we demonstrate that the haematopoietic defect in DNA-ligase-I-deficient embryos is a quantitative deficiency relating to reduced proliferation rather than a qualitative block in any haematopoietic lineage. DNA ligase I null fibroblasts from Lig1 mutant embryos showed an accumulation of DNA replication intermediates and increased genome instability. In the absence of a demonstrable deficiency in DNA repair we postulate that, unusually, genome instability may result directly from the DNA replication defect. Lig1null mouse cells performed better in the survival and replication assays than a human LIG1 point mutant, and we suggest that the complete absence of DNA ligase I may make it easier for another ligase to compensate for DNA ligase I deficiency.

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Global detection of DNA repair outcomes induced by CRISPR-Cas9

10.1101/2021.02.15.431335 ◽

2021 ◽

Author(s):

Mengzhu Liu ◽

Weiwei Zhang ◽

Changchang Xin ◽

Jianhang Yin ◽

Yafang Shang ◽

...

Keyword(s):

Dna Repair ◽

Genome Editing ◽

Target Genes ◽

Chromosomal Translocations ◽

Dna Breaks ◽

Dsb Repair ◽

Large Deletions ◽

Mouse Cells ◽

Repair Pathways ◽

Cellular Dna

AbstractCRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, plasmid integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

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