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. 2017 Aug 10;45(18):10614–10633. doi: 10.1093/nar/gkx715

Figure 2.

Figure 2.

H2AX deficiency reduces the efficiency and deletion length in mNHEJ-mediated CRISPR/Cas9 genome editing. (A) Percentage of gRNA/Cas9-induced GFP+ cells from H2AX+/+ and H2AX–/– sGEJ reporter cells. Three gRNAs (g2–1, g2–2 and g2–3) targeting the regions surrounding the second I-SceI site are indicated. Values are the mean ± S.D. of three independent experiments, each in triplicates. Student paired t-test between ‘H2AX+/+’ and ‘H2AX–/–’: P = 0.03 for g2–1; P = 0.047 for g2–2; P = 0.007 for g2–3. (B) Percentage of g2–2/Cas9-induced GFP+ cells from H2AX–/– sGEJ reporter cells stably transfected with expression plasmids for HA-tagged mouse H2AX and its mutant S139A as well as empty vector control (EV). Values are the mean ± S.D. of three independent experiments, each in triplicates. Student paired t-test between ‘EV’ and ‘H2AX’: P = 0.0008; between ‘EV’ and ‘S139A’: not significant (NS). HA-tagged H2AX and S139A detected by anti-HA antibody are indicated under the bar chart with β-actin as the loading control. (C) Deletion distributions of ‘Del’ events in g2–2/Cas9-induced mNHEJ between H2AX+/+ and H2AX–/– sGEJ reporter ES cells demonstrate a shift toward shorter deletions in H2AX–/– cells. Each blue or red dot represents 20 reads. ****P < 0.0001 (two-tailed Mann–Whitney test). (D) Frequency of deletions with different deletion length in ‘Del’ events of g2–2/Cas9-induced mNHEJ between H2AX+/+ and H2AX–/– sGEJ reporter cells. The NHEJ events were grouped into 1–3 bp, >3 bp (excluding SSA or MMEJ) and 34 bp (SSA or MMEJ events) according to the deletion length. The combined reads and frequencies were summarized in inset with P values from a χ2 test indicated. (E) The relative efficiency of Cas9-induced mNHEJ at three different sites of endogenous ROSA26 locus in H2AX+/+ and H2AX–/– mouse ES cells. Three gRNAs (gR26–1, gR26–2 and gR26–3) targeting three different sites of endogenous ROSA26 locus are indicated. Cas9-induced mNHEJ was identified by Illumina sequencing, and the relative efficiency was calculated as ratios of mNHEJ reads to total reads and normalized by transfection efficiency. Student's paired t-test between ‘H2AX+/+’ and ‘H2AX–/–’: P = 0.004 for gR26–1; P = 0.003 for gR26–2; P= 0.019 for gR26–3. (F) Frequency of deletions with different deletion length in ‘Del’ events of gR26–3/Cas9-induced mNHEJ at the ROSA26 locus between H2AX+/+ and H2AX–/– mouse ES cells. The NHEJ events were grouped into deletions of 1–2 bp, deletions over 2 bp (>2 bp) and deletions of 6 bp (MMEJ). The combined reads and frequencies were summarized in inset with P values from a χ2 test indicated.