The genomic revolution has arrived, and the research community is developing new tools for determining how genotype influences phenotype. Using genome engineering, scientists can inactivate and replace genes with improved site-specificity. The continuous improvement of nuclease technologies (zinc finger nucleases, TALENS) and the recent development of CRISPR/Cas systems have expanded the toolbox of genome editing tools and technologies. Read on to find the plasmids you need to start engineering:
Addgene’s Genome Engineering Toolbox
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) systems are the newest tools in the genome engineering collection. Discovered in bacteria, the CRISPR/Cas system acts as the bacteria’s defense mechanism against foreign DNA. The CRISPR systems developed for genome engineering in eukaryotic cells require two parts: 1) the Cas9 nuclease, which induces the double strand break and 2) the guide RNA (gRNA), a synthetic, customizable element that binds, activates and targets the Cas9 nuclease.
The system is easy to customize for many genome editing applications. Synthetic gRNA can be designed to target virtually any 20 base pairs of the form N20NGG by simply inserting the target sequence into a gRNA expression plasmid. Additionally, the Cas9 nuclease has been optimized for use in a variety of species, including human, zebrafish, yeast and more, with little need for further modification. A number of different gRNA expression plasmids and codon-optimized Cas9 plasmids are currently available through Addgene, allowing for selection of reagents based on your genome editing experiments.
The CRISPR/Cas system is not only streamlining the number of reagents needed for genome engineering (just 2 plasmids), but also simplifies the engineering requirements, with no need for enzyme engineering of the nuclease. As more labs utilize the technique for mammalian genome modification, we will see how the technology compares to the other tools available (described below).
Transcription activator-like effector nuclease (TALEN) systems are a fusion of TALEs derived from the Xanthomonas spp. to a restriction endonuclease FokI. By modifying the amino acid repeats in the TALEs, users can customize TALEN systems to specifically bind target DNA and induce cleavage by the nuclease between the two distinct TAL array binding sites.
An advantage of TALEN technology is that each individual TALE repeat specifically recognizes a single base pair. Thus, connecting numerous TALE repeats allows the user to create custom, site-specific DNA binding proteins. Similar to the zinc finger technology which came before it (described in the next section), TALEN systems require the binding of TALE repeats at two separate binding sites, which then permits dimerization and activation of the FokI nuclease.
The TALEN technology continues to be a popular option for site-specific genome engineering applications. Currently there are a variety of plasmid kits to choose from in Addgene’s repository. These kits allow for easy assembly of custom TALEN arrays using various cloning techniques and protocols. The technologies available include the Golden Gate cloning method, assembly by serial ligation, and assembly via ligation-independent cloning. These TALEN kits have been validated in a variety of cell types, including human, zebrafish, and more.
Zinc Finger Nucleases
Zinc finger nuclease (ZFN) technology utilizes a FokI nuclease as the DNA-cleavage domain and binds DNA by engineered Cys2His2 zinc fingers. Specific zinc fingers recognize different nucleotide triplets and when joined in arrays they can impart sequence specificity of 9 to 18 base pairs. Upon recognition of the DNA sequence on opposite sides of the DNA strand by two zinc fingers, dimerization of two FokI nuclease domains occurs. This activated nuclease can then introduce a double stranded break between the two zinc finger binding sites, which enables subsequent modification of the genome.
Methods have been developed that facilitate easy construction of customized, site-specific zinc finger arrays. These techniques include the Oligomerized Pool ENgineering (OPEN) method and the modular assembly method. ZFNs have been successfully used for genome engineering for over 10 years and have begun to make their way into clinical trials.
Which tool is the right one for you?
Zinc finger nucleases have been around the longest and have been characterized the best of all three technologies. That said, many ZFN users are migrating over to the TALEN system due to the newer system’s increased flexibility. If you are trying to choose between TALEN and CRISPR technology, your decision might ultimately be dependent upon your application. CRISPRs have been shown to cut their target sites significantly more efficiently than TALENs, but unlike TALENS, CRISPRs are more prone to cutting offsite targets. Both technologies are evolving rapidly, and at some point one technology may come out on top as both highly efficient and highly accurate. For now, genome engineers can enjoy the large selection of tools in their toolbox and know that modifying a genome has quickly moved from being a massive project to a relatively quick step in the overall scope of their experiments.
- Gaj T, et al. Trends Biotechnol. 2013 May 8. pii: S0167-7799(13)00087-5.
- Ramalingam S, et al. Genome Biol. 2013 Feb 26;14(2):107.
- Carlson DF, et al. Mol Ther Nucleic Acids. 2012 Jan 24;1:e3.
For further information, visit: http://www.addgene.org/Genome_Engineering/