Fig 2: Examination of PARP1 protein status after gene editing triggered by DSBs versus SSBs. (A) Confocal microscopy analysis of HeLa cells expressing untagged and EGFP-tagged PARP1. Confocal microscopy of EGFP::PARP1+ and EGFP::PARP1- cells confirming co-localization of PARP1 and EGFP in the nuclei of the former cell populations engineered by in trans paired nicking or standard gene editing. Nuclei were counterstained with DAPI. Unedited HeLa cells served as negative controls. Specimens of EGFP::PARP1- cells not incubated with the primary PARP1-specific antibody (-1st Ab) provided for an additional staining control. (B) Western blot analysis of HeLa cells expressing untagged and EGFP-tagged PARP1. Western blotting of EGFP::PARP1+ and EGFP::PARP1- cells exposing a striking reduction in the amounts of endogenous PARP1 antigen exclusively in EGFP::PARP1+ cells generated through standard DSB-dependent gene editing (open arrowhead). Properly sized EGFP::PARP1 fusion products were detected in both EGFP::PARP1+ cell populations (solid arrowhead). Unedited HeLa cells served as negative controls. a/ß Tubulin antigens served as internal protein loading controls.

Fig 3: Homology-directed PARP1 gene editing based on cleaving or nicking CRISPR complexes. (A) Diagrams of PARP1 and PARP1-tailored gene editing tools. The gRNAPARP1 target site (TS) is indicated by the horizontal arrow and boxed nucleotides (PAM). The vertical dashed line marks the SpCas9:gRNAPARP1 cleaving position. The N-terminal PARP1 amino acids are drawn next to their respective codons. The donor constructs pDonorPARP1 and pDonorPARP1.TS have as targeting module PARP1 sequences (‘arms of homology’) flanking a EGFP tag. The latter construct has, in addition, TS sequences flanking the targeting module. (B) Quantification of genetically modified human cells. Flow cytometry of HeLa cell cultures co-transfected with the indicated plasmids. Data are presented as mean ± S.D. of three independent biological replicates. Significance between the indicated datasets was calculated with one-way ANOVA followed by Tukey's test for multiple comparisons; *P< 0.05; **P< 0.01. (C) Molecular characterization of human cells genetically modified through standard versus in trans paired nicking gene editing at PARP1. Top panel, Junction PCR assay for assessing PARP1 gene tagging. Amplicons diagnostic for HDR-derived centromeric and telomeric junctions between exogenous DNA and PARP1 (jC and jT, respectively) are depicted. Amplicons specific for EGFP served as internal controls (EGFP). Bottom panel, Junction PCR analysis on genomic DNA from EGFP+ HeLa cell clones retrieved from cultures co-transfected with pCas9, pDonorPARP1 and pgRNAPARP1 (Standard setting) or with pCas9D10A, pDonorPARP1.TS and pgRNAPARP1 (In trans paired nicking setting). H2O, PCR sample containing nuclease-free water instead of genomic DNA. Lanes M, GeneRuler DNA Ladder Mix molecular weight marker. (D) Competition experiment involving unedited and PARP1 edited cells. Long-term cultures of HeLa cells expressing EGFP-tagged PARP1 mixed with unedited cells were monitored by flow cytometry. Green and magenta lines, EGFP+ cells generated by in trans paired nicking and standard gene editing, respectively.