Impact of demethylating treatment on targeted mutagenesis (TM) and homologous gene targeting (HGT) frequencies induced by engineered meganucleases in 293-H cells.

<p>(<b>A</b>) TM frequency was determined from cells grown with or without 0.2 µM or 1 µM 5-aza-dC and transfected with the engineered meganuclease (MN) XPCm, or RAG1 m and CAPNS1 m, two meganucleases targeting DNA sequences that lack methylated CpG. (<b>B</b>) Distribution of TM events in methylated (white) and unmethylated (black) sequences from cells transfected with XPCm with and without 5-aza-dC. (<b>C</b>) HGT frequency was determined from cells grown with 0.2 µM (+) or without (−) 5-aza-dC and co-transfected with the DNA repair matrix (RM) and the XPCm engineered meganuclease (+) or empty vector (−).</p

Homologous gene targeting (HGT) induced by the TALEN™ (XPCT1) in XP4PA cells.

<p>(<b>A</b>) Western blot performed on protein extracts from cells transfected with XPCT1 (+) or empty vector (−). Each monomer, XPCT1R and XPCT1L was tagged with S-tag and HA-tag, respectively. (<b>B</b>) HGT frequency was determined from XP4PA cells transfected with XPCT1 (+) or non-related TALEN™ (−) in the presence of the DNA correction matrix described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078678#pone-0078678-g002" target="_blank">Figure 2</a>. The transfected cells were seeded at a density of 20 cells/well or lower, enabling the formation of individual clones.</p

Phenotypic correction of XP4PA cells.

<p>(<b>A</b>) Western blot performed on protein extracts from clones derived from transfection with the meganuclease XPCm in the presence of demethylating treatment (left panel) or from transfection with the TALEN™ XPCT1 (right panel). XPC expression of corrected clones (Corr) was compared to negative controls, XP4PA (1), to uncorrected ΔTG clones (3), and to a positive control MRC5, proficient for XPC (2). In the left panel, an additional band is revealed by the XPC antibody. This band is most probably due to the non-specific binding of the antibody. Furthermore, this could be heightened by the 5-aza-dC treatment, as the band seems to appear only in treated samples. (<b>B</b>) UV-C survival assay on clones derived from gene correction experiment using XPCm (left panel) or using XPCT1 (right panel). The percentage of cell survival after exposure to UV-C of XPC corrected clones (closed triangle and lozenge)) was compared to two negative controls, XP4PA and uncorrected ΔTG clone (open triangle and lozenge, respectively) and one positive control MRC5 (closed square).</p

Efficacy of XPCm in XP4PA cells after 5-aza-dC treatment.

<p>(<b>A</b>) Chromatogram showing the impact of 5-aza-dC treatment on methylating status of the XPCt. Cells were grown with 0.2 µM (+) or without (−) 5-aza-dC and transfected with empty vector under the same conditions as in TM or HGT expriments. While the CpGs present in XPCt was fully methylated under non-treated conditions, the 5-aza-dC treatment induced partial demethylation as shown by the presence of a double peak. (<b>B</b>) TM frequency was determined from XP4PA cells grown with 0.2 µM (+) or without (−) 5-aza-dC and transfected with XPCm (+) or empty vector (−). (<b>C</b>) Design of the DNA correction matrix used for HGT experiments, which was composed of two arms of 1,579 bp and 1,830 bp, homologous to the <i>XPC</i> sequences and separated by the underlined meganuclease-recognizing site (part of the normal wild type sequence of <i>XPC</i>). The DNA sequence that was recognized by the meganuclease was modified by producing silent mutations (in red letters) to avoid any cleavage of the matrix by XPCm. (<b>D</b>) Sequencing of HGT-PCR products from one corrected population (CP). These sequences were compared to the sequences obtained in the MRC5 cell proficient for XPC (+) and in the parental cell line XP4PA carring the TG deletion (−).</p