Identification of pathogenic variants in cancer genes using base editing screens with editing efficiency correction.

BACKGROUND: Millions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens. RESULTS: To improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons of BRCA1 and BRCA2 genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants for BRCA1/2, including 151 variants in the noncoding part of the genes such as the 5' untranslated regions. Many of them are identified in cancer patients and are reported as "benign/likely benign" or "variants of uncertain significance" by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer. CONCLUSIONS: Our results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.

Genome-wide 5-Hydroxymethylcytosine Profiling Analysis Identifies MAP7D1 as A Novel Regulator of Lymph Node Metastasis in Breast Cancer.

Although DNA 5-hydroxymethylcytosine (5hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genome-wide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated 5hmC signatures, and it is found that increased levels of DNA 5hmC and gene expression of MAP7D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic 5hmC profiles during lymph node metastasis suggest a link between DNA 5hmC and lymph node metastasis. Meanwhile, the role of MAP7D1 in breast cancer progression suggests that the metastasis-associated 5hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.