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.
CRISPR has quickly become the preferred system for genome engineering due to its simplicity, as it requires only Cas9 and a guide RNA (gRNA). Choosing the correct method to deliver both Cas9 and gRNAs to your target cells is absolutely critical as failure to adequately express either component will result in a failed experiment. In our previous blog post entitled “CRISPR 101 - Mammalian Expression Systems and Delivery Methods” we provided a general overview of the most common ways in which you can deliver Cas9 and gRNAs to your target cells and discussed a few key advantages and disadvantages of each method. In this blog post, we will go into greater detail about why and how Cas9/gRNA Ribonucleoprotein complexes (Cas9 RNPs) are being used for genome engineering experiments and provide a general framework for getting started with Cas9 RNPs in your research.
At their most basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post, we hope you will be caught up on much of the major CRISPR lingo and will be able to describe the functions of the various CRISPR/Cas9 components. Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements of these different systems can vary (for example, read our posts on Cpf1 and eSpCas9/SpCas9-HF1 for some of the interesting properties of these exciting new nuclease tools).
Topics: CRISPR 101
The advent of CRISPR/Cas9 made it easier than ever to efficiently make precise, targeted genome modifications. Cas9 has been modified to enable researchers to knock out, activate, repress or even image your favorite gene. But, with such a wide variety of Cas9-based reagents available, how do you choose which Cas9 is right for your particular experiment? This blog post will help familiarize you with the wide array of Cas9s available through Addgene’s repository and make it easy to select the Cas9 reagent that is right for you.
The first thing to do in any CRISPR experiment is identify what, exactly, you are trying to do. Are you trying to permanently knock-out a gene in a cell type or organism? Are you trying to reduce expression of a particular gene without permanently modifying the genome? Does it make more sense to try and activate at a particular locus? What about modifying the epigenome at a particular location? As you might expect, the answer to this question will substantially affect your decisions about which Cas9 you need for your experiment. Below is a brief summary of a few of the common genetic manipulations one can carry out using Cas9 and the specific Cas9s that can be used for each.
We recently wrote about how Cpf1 is pushing CRISPR to new horizons, but Cas9 definitely still has a few tricks up its sleeve. Diverse genomes and genomic targets require a variety of tools to engineer them effectively. Read on to learn how a variety of natural and engineered forms of Cas9 can be used to expand CRISPR's reach to new genomic loci similarly to Cpf1. If you'd like to learn more, you should continue to our newly re-vamped CRISPR guide to keep up to date on all things CRISPR.