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.
This post was contributed by guest blogger, member of the Addgene Advisory Board, and Associate Director of the Genetic Perturbation Platform at the Broad Institute, John Doench.
A genetic screening project can be a tremendous undertaking, producing a wall of results that can only be described as bigly. But such a project should not be undertaken lightly. Whether executed in arrayed or pooled format there are of course materials costs, regardless of who is paying for them. More importantly, there’s the opportunity cost of your time; an investment of months of your life that may end with little more than an Excel spreadsheet of random numbers that’ll leave you, well, #sad.
This post was contributed by guest bloggers Marcelle Tuttle and Alex Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering.
Background on Cas9 Activators
CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been most widely used to create specific edits in DNA, there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow for the upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1).
When we talk about CRISPR applications, one negative always comes up: the low editing efficiency of homology-directed repair (HDR). Compared to the random process of non-homologous end joining, HDR occurs at a relatively low frequency, and in nondividing cells, this pathway is further downregulated. Like all CRISPR applications that use wild-type Cas9, editing by HDR also has some potential for off-target cleavage even when gRNAs are well designed. Rather than try to improve HDR, Addgene depositor David Liu’s lab created new Cas9 fusion proteins that act as “single base editors.” These fusions contain dCas9 or Cas9 nickase and the rat cytidine deaminase APOBEC1, which can convert cytosine to uracil without cutting DNA. Uracil is subsequently converted to thymine through DNA replication or repair. Komor et al. estimate that hundreds of genetic diseases could be good targets for base editing therapy, not to mention the potential basic and preclinical research applications. Read on to learn about this new way to make point mutations using CRISPR without double-stranded breaks.