Biological Sciences

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

DNA methylation is a key epigenetic mechanism underlying many biological processes, and its aberrant regulation has been tightly associated with various human diseases. Precise manipulation of DNA methylation holds the promise to advance our understanding of this critical mechanism and to develop novel therapeutic methods. Previously, we were only able to alter genome-wide DNA methylation by treating with small molecules (e.g., 5-Aza-2-deoxycytidine) or perturbing relevant genes (e.g., DNA methyltransferase) targetlessly, which makes it challenging to investigate the functional significance of this epigenetic mark at specific genomic loci. By fusing the catalytic domain of a key enzyme in the DNA demethylation process (Ten-eleven translocation dioxygenases 1, Tet1) with a reprogrammable sequence-specific DNA-targeting molecular protein, dCas9, we developed a DNA methylation editing tool (dCas9-Tet1) to demethylate specific genomic loci in a targeted manner. This dCas9-Tet1 system allows us to study the role of DNA methylation at almost any given loci with only the replacement of a single-guide RNA. Here, we describe a protocol that enables modular and scalable manipulation of DNA methylation at specific genomic loci in various cell cultures with high efficiency and specificity using the dCas9-Tet1 system.

The CTC1-STN1-TEN1 (CST) complex is a single-strand DNA-binding protein complex that plays an important role in genome maintenance in various model eukaryotes. Dysfunction of CST is the underlying cause of the rare genetic disorder known as Coats plus disease. In addition, down regulation of STN1 promotes colorectal cancer development in mice. While prior studies have utilized RNAi to knock down CST components in mammalian cells, this approach is associated with off-target effects. Attempts to employ CRISPR/Cas9-based knockout of CST components in somatic cell lines have been unsuccessful due to CST's indispensable role in DNA replication and cell proliferation. To address these challenges, we outline a novel approach utilizing a Cre-loxP-based conditional knockout in mouse embryonic fibroblasts (MEFs). This method offers an alternative means to investigate the function and characteristics of the CST complex in mammalian systems, potentially shedding new light on its roles in genome maintenance.