Raw data of Affymetrix ATH1 GeneChip and RNA-seq data can be found in NCBI as described above.Source data related to LithoGraphX analysis (Fig. 3c, d; Supplementary Fig. 11) and ANOVA analysis (Fig. 4d; Supplementary Figs. 8e, 11, 12, 13) can be found in Supplementary Datasets. The data that support the findings of this study are available from the corresponding author upon request.
BackgroundMany applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.ResultsIn the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.ConclusionsNHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1518-x) contains supplementary material, which is available to authorized users.
Metastasis is one of the typical features of malignancy that significantly increases cancer-related mortality. Recent studies have shown that epithelial–mesenchymal transition (EMT) is closely related to the invasion and migration of cancer cells. Grainyhead-like 2 (Grhl2), a transcription factor, has been reported to be associated with several tumor processes including EMT. In the previous study, we have reported that Grhl2 functioned as a tumor suppressor in proliferation and apoptosis of gastric cancer. Here we aim to explore the effects of Grhl2 on invasion and migration of gastric cancer and further clarify its possible underlying mechanisms. As a result, in both SGC7901 and MKN45 cells, Grhl2 overexpression significantly inhibited the ability of invasion and migration. In addition, preliminary experiments showed that Grhl2 reduces the protein expression of matrix metalloproteinase-2, -7 and -9 (MMP-2, MMP-7 and MMP-9). Most importantly, Grhl2 antagonizes transforming growth factor-β (TGFβ)-induced EMT, and inhibition of TGFβ signaling pathways can restore Grhl2 expression. Finally, the results of subcutaneous xenograft model indicated that Grhl2 suppresses the growth of gastric cancer and reverses EMT process in vivo. Meanwhile, the metastatic tumor model further confirmed the inhibition of Grhl2 on metastasis of gastric cancer. Taken together, our findings proved that Grhl2, functioned as a tumor suppressor, reduces the invasion and migration through inhibition of TGFβ-induced EMT in gastric cancer.
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