Epigenetic modifications are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose!
This post was contributed by guest bloggers Alissa Lance-Byrne and Alex Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering.
CRISPR/Cas9 technology has revolutionized the fields of molecular biology and bioengineering, as it has facilitated the development of a simple and scalable means of making targeted genetic edits. Cas9 is a DNA binding protein that can be directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to either inactivate a gene or introduce a desired genetic alteration.
Numbers in the large colored circles are rough approximations of the total number of CRISPR plasmids for that particular organism available at Addgene. Percentages represent the fraction of that total with the indicated function.
One huge reason CRISPR has become such a popular genome editing tool is its developers’ willingness to make their CRISPR technologies available to the academic research community. At Addgene, we’ve helped distribute many of these technologies in plasmid form and are proud to have facilitated their fast adoption. However, in many cases the plasmids themselves are only the starting point for the production of viruses used to deliver CRISPR components to cells or organisms under study. In the past we’ve left the arduous task of virus production to individual labs, but now we’re very excited to provide ready-to-use CRISPR lentiviral preps to researchers across the globe.
CRISPR has taken the genome engineering world by storm owing to its ease of use and utility in a wide variety of organisms. While much of current CRISPR research focuses on its potential applications for human medicine (1), the potential of CRISPR for genome engineering in plants is also being realized. There are a variety of reasons to consider using genome editing to change the genetic code of plants, including the development of crops with longer shelf life and the development of disease-resistant crops to increase agricultural yield (2,3). While it is certainly possible to select for desirable traits using traditional plant breeding approaches, these techniques are cumbersome, often requiring several rounds of selection to isolate plants with the phenotype of interest. Genome engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already been used to engineer the genome of many plant species, including commonly used model organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn, tomato, wheat, mushroom, and rice (4). Despite the almost universal functionality of the CRISPR system in most organisms, some plant-specific changes to CRISPR components are necessary to enable genome editing in plant cells.
This post was contributed by guest blogger Søren Hough, the Head Science Writer at Desktop Genetics.One of the most important steps in the CRISPR experimental process is validating edits. Regardless of which CRISPR genome editing system you use, there remains a chance that the observed phenotype was caused by an off-target mutation and not an edit in the target gene.
The validation process, also known as CRISPR genotyping, is critical to demonstrating causal relationships between genotype and assayed phenotype. Verifying these connections can help alleviate the reproducibility crisis in biology. It is key to address these concerns as CRISPR use grows across the life sciences and to establish standardized validation techniques for academia, industry, and especially the clinic.