Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation.

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ES cells contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly due to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hES cells, we find iPS cells tend to form large-scale (100 kb-1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which display incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (~80%) with aberrant iPS-ES non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute the epigenetic variation of iPSCs and iPSC-hESC differences.

The discovery of circulating fetal nucleic acids is a great step on the way of developing non-invasive prenatal diagnosis (NIPD) for genetic disorders. Here, we briefly discuss the current applications of circulating fetal nucleic acids in genetic testing for different kinds of hereditary diseases with an emphasis on using circulating cell-free fetal DNA in diagnosis of monogenic disorders. As the genetic skin disorders impair the quality of life at different levels, we next discuss some ethical issues in NIPD for genetic skin diseases of various severities and in particular, the responsibility of doctors and parents, respectively, in the prenatal genetic testing.

Despite dedicated research in precision medicine, identification of therapeutic strategies for oncogenic drivers remains a major challenge as many mutations are difficult to target directly. Synthetic lethality-based target prediction becomes a powerful approach for drug discovery. We previously developed a computational platform, Mining Synthetic Lethals (MiSL), that can output a list of possible synthetic lethal partners of a specific gene when fed with primary human tumor genomic and transcriptomic data. The principal logic of the MiSL algorithm is based on the Boolean implication that the SL partners of a mutation will never be deleted in the presence of the mutation in the primary tumors. High frequency of KRAS mutation in lung and colon cancers makes it an important therapeutic target. While drugs directly targeting KRAS are yet to be fully developed, we applied the principle of synthetic lethality using the MiSL platform to predict potential KRAS specific therapeutic targets. We identified 556 putative SL partners of KRAS mutations combining lung and colon cancers. To efficiently test all predictions, we used customized shRNA and CRISPR lentiviral libraries to validate all the predicted SL interactions for KRAS in colon cancer cell lines HT29 (KRAS WT) and SW620 (KRAS G12V mutant). Eight shRNAs and four sgRNAs were designed for each gene in the shRNA and CRISPR libraries. The shRNAs and sgRNAs expressed at baseline and at the end of each screen were analyzed by barcode sequencing. Synthetic lethal interactions with KRAS were determined by identifying individual sh/sgRNAs whose mean values at the end relative to the start of the screen were significantly reduced in KRAS Mut cell lines compared to WT. We also compared the results of the in vitro to an in vivo screen of the focused libraries in human tumors xenografted into mice using the same cell lines. We compared the top 50 SL candidates from each screen as ranked by MAGeCK (Model-based Genome-wide Analysis of CRISPR/Cas9 KO) and found that only 19 were shared between the in vitro and in vivo screens. Further validation of several top SL candidates coming from both in vitro and in vivo screens was performed by testing individual shRNAs/sgRNAs for growth inhibition in KRAS Mut (SW620) vs. KRAS WT (HT29) cell lines in vitro and in vivo. Individual in vitro CRISPR/Cas9 knock out of two of the candidate SL partners reduced cell viability by 50% and 57% respectively in KRAS mutant cells compared to KRAS WT cells. Furthermore, induced individual KD or KO of four SL partners resulted in inhibition of tumor growth by 84%, 68%, 95% and 60%, respectively. In summary, MiSL has shown to be a powerful platform for discovering novel targets for specified cancer types. This study has identified and validated several genes as promising targets of KRAS dependent colon cancer for further therapeutic investigation. Citation Format: He Gong, Claire Repellin, Puja Patel, Merrill Knapp, Mingze He, Jun Li, Lisa Wu, Xiaohe Liu, Subarna Sinha, Lidia Sambucetti. Validation of computationally predicted synthetic lethal interactions for KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB108.