Journal: eLife

Article Title: PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

doi: 10.7554/eLife.69504

Figure Lengend Snippet:

Article Snippet: Plasmids acquired from the non-profit plasmid distribution service Addgene were the following: pCMV-PE2 (#132775) , pAT9624-BEAR-cloning (#162986), pAT9651-BEAR-GFP (#162989), pAT9752-BEAR-mScarlet (#162991), pAT9658-sgRNA-mCherry (#162987), pAT9679-sgRNA-BFP (#162988) , pX330-Flag-wtSpCas9-H840A (#80453), pX330-Flag-dSpCas9 (#92113), and pX330-Flag-wtSpCas9 (#92353) ( ).

Techniques: Recombinant, Sequencing

Journal: PLoS Pathogens

Article Title: Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

doi: 10.1371/journal.ppat.1010275

Figure Lengend Snippet: (A-B) Percentage of HFK WT and p300 KO cells with (A) colocalized RPA70 (green) and pDNA-PKcs (red) foci or (B) RAD51 (green) and pDNA-PKcs (red) foci over a 32-hour time course following zeocin exposure (10 μg/mL, 10 min). (C-D) Percentage of CCS1477 (1 μM) or DMSO treated HFK LXSN cells that contained colocalized (C) RPA70 (green) and pDNA-PKcs (red) or (D) RAD51 (green) and pDNA-PKcs (red) foci following zeocin treatment. (E-F) Percentage of primary HFK cells treated with CCS1477 or DMSO that contained (E) colocalized RPA70 and pDNA-PKcs foci 16-hours following zeocin treatment or (F) RAD51 and pDNA-PKcs foci 24-hours following zeocin treatment. All values are represented as mean ± standard error. The statistical significance of differences between cell lines or treatments were determined using Student’s t-test. * indicates significant difference between two groups (A-D) or LXSN and 8E6 (E-F) with same treatment (p< 0.05). # indicates p < 0.05 between control (0 h) and zeocin treated group within each cell line. At least 150 cells were counted over three independent experiments.

Article Snippet: CCS1477 (CT-CCS1477, Chemietek) was used to inhibit p300 activity (1 μM). sgRNA/CAS9 plasmids (#136938, Addgene) were used to generate a DSB for next-generation sequencing.

Techniques: Control

Journal: PLoS Pathogens

Article Title: Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

doi: 10.1371/journal.ppat.1010275

Figure Lengend Snippet: (A-B) Percentage of HFK WT and HFK p300 KO cells with RAD51 staining in G1 as determined by (A) Cyclin E staining or (B) flow cytometry after zeocin treatment (10 μg/mL, 10 min). (C-D) Percentage of HFK LXSN cells treated with DMSO or CCS1477 (1 μM) that had RAD51 staining in G1 as determined by (C) Cyclin E staining or (D) flow cytometry after zeocin treatment. (E-F) Percentage of primary HFK LXSN cells treated with DMSO or CCS1477 that had RAD51 staining in G1 as determined by (E) Cyclin E staining or (F) flow cytometry after zeocin treatment. All values are represented as mean ± standard error. The statistical significance of differences between cell lines or treatments were determined using Student’s t-test. * indicates significant difference between two groups (A-D) or LXSN and 8E6 (E-F) with same treatment (p< 0.05). # indicates p < 0.05 between control (solvent) and treated group within each cell line. At least 150 cells were counted over three independent experiments for microscopy. Twenty thousand cells were counted for each of three independent flow cytometry experiments.

Article Snippet: CCS1477 (CT-CCS1477, Chemietek) was used to inhibit p300 activity (1 μM). sgRNA/CAS9 plasmids (#136938, Addgene) were used to generate a DSB for next-generation sequencing.

Techniques: Staining, Flow Cytometry, Control, Solvent, Microscopy

Journal: PLoS Pathogens

Article Title: Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

doi: 10.1371/journal.ppat.1010275

Figure Lengend Snippet: (i) DSB occurs in a cell in G1 phase that is expressing 8E6. (ii) NHEJ initiates. DNA-PKcs is recruited to the lesion where it is auto-phosphorylated. (iii) NHEJ stalls due to 8E6 mediated p300 degradation. This leaves unresolved pDNA-PKcs repair complexes . (iv) HR initiates at the site of failed NHEJ. The MRN complex, CtIP, and EXO1 resect one strand of DNA near the lesion, producing single stranded DNA . RPA complexes coat and stabilize the resulting single stranded DNA . RPA70 foci colocalize with pDNA-PKcs indicating that HR-mediated DNA resection occurs after NHEJ fails. Then, RAD51 is recruited to the break site. (v) HR cannot be complete due to the lack of a homologous template and/or 8E6-mediated inhibition of HR . This causes deletions due to antagonist DNA end process by NHEJ and HR and/or by failure to complete HR after resection. (vi) The failure of NHEJ causes cells to use tertiary DSB repair pathways to fix the lesion. (vii) This alternative repair pathway (e.g., Alt-EJ) is error prone and increases other types of mutations (such as SNPs).

Article Snippet: CCS1477 (CT-CCS1477, Chemietek) was used to inhibit p300 activity (1 μM). sgRNA/CAS9 plasmids (#136938, Addgene) were used to generate a DSB for next-generation sequencing.

Techniques: Expressing, Inhibition