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 to use and when not to use Cas9 activators
One of the best uses for Cas9 Activators is in genetic screening. gRNAs targeting every gene in the human genome for example, can be made easily and cheaply using oligo library synthesis. Prior to Cas9 activators, similar tools were made using other DNA binding proteins such as zinc fingers (ZF) and TAL effectors (TALE). Unlike these constructs, however, Cas9 allows you to easily change the sequence targeted by the activator by simply providing a new gRNA rather than engineering an entirely new protein. This makes it much cheaper to use Cas9 activators.
cDNA libraries, which consist of plasmids that over-express coding sequences from a given cell type or organism, have been used in a similar manner to Cas9 activators. However, these can be difficult to construct and deliver when compared to gRNAs. Additionaly, cDNAs cannot be used to study in cis regulation and also suffer from an inability to easily deliver the appropriate isoform(s) of a given gene, as, many times, the isoform(s) common to a particular cell type are unknown or not readily available. By activating from the native context of the gene, Cas9 activators efficiently solve these problems.
While Cas9 activators can be enormously powerful tools, they’re not a good fit for every application. For example, Cas9 activators are particularly bad for experiments wherein you only want to activate a single gene; as there are several factors that may prevent your gene from being up-regulated. For instance, generally, the more highly expressed a gene is under native conditions, the less activation you can achieve using a Cas9 activator; your gene of interest might already be hitting an upper bound of activation that current Cas9 systems cannot help you pass. In addition, activation experiments often require quite a bit of tuning before you know your system is working as expected. Finally, for each gene you want to activate, you should also be ready to test three or four guides directed towards that gene as there can be a large difference in guide potency.
All of the above concerns with Cas9 activators are, for the most part, less of an issue in the setting of large scale genome-wide screens as the only consequence of a gene not being properly up-regulated in this setting is a potentially missed hit rather than complete experimental failure. Thus, in cases where users have a single gene they want to activate, we would recommend using a cDNA overexpression vector rather than going through all the troubleshooting required for Cas9-based activation.
Finally, we would like to point out that, while Cas9 activators are very useful for in vitro experiments, the technology is not exactly there yet for in vivo experiments. Most Cas9 activators are simply too large to fit all components into the most promising delivery vector, Adeno-associated virus (AAV). In the future, however, smaller Cas9 orthologs and transcription factor components could lead to an activator small enough to fit into the AAV chassis and yet still retain the ability to potently induce targeted gene expression.
Which Cas9 activator should I use?
There are a wide variety of activators you can use for your experiments. We have found that SAM (1), Suntag (2, 3), and VPR (4) are good choices across multiple cell lines (HEK293T, MCF7, U2-OS, Hela, N2A, 3T3) and organisms (5). Our general advice, however, is to use whichever activator is most accessible to you and which you are most familiar with.
Worry less about off targets
Unlike Cas9 cutting activity, off target effects are generally not regarded as being a large problem for Cas9 activators. This is believed to be true given the results of previous RNA-seq experiments (1, 4, 5) along with a belief that the odds are very low that Cas9 would have an off-target that lands in the promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides by putting the promoter of the gene into a gRNA finder such as WU-CRISPR (6) or our lab’s sgRNA scorer 1.0 (7) and picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the guides to a region less than 200 bp upstream of TSS for best results but up to 400 bp works reasonably well.
Best of luck on your experiments!
Many thanks to our guest bloggers Marcelle Tuttle and Alex Chavez!
Marcelle Tuttle is a medical student at Tufts University and former researcher at the Wyss Institute for Biologically Inspired Engineering.
Alejandro (Alex) Chavez is a member of the Addgene Scientific Advisory Board with a particular interest in generating Cas9 based tools to enable facile control of DNA and RNA.
2.Tanenbaum, Marvin E., et al. "A protein-tagging system for signal amplification in gene expression and fluorescence imaging." Cell 159.3 (2014): 635-646. PubMed PMID: 25307933. PubMed Central PMCID: PMC4252608.
5. Chavez, Alejandro, et al. "Comparison of Cas9 activators in multiple species."Nature methods (2016). PubMed PMID: 27214048.
6. Wong, Nathan, Weijun Liu, and Xiaowei Wang. "WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system." Genome biology16.1 (2015): 1. PubMed PMID: 26521937. PubMed Central PMCID: PMC4629399.
7. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach." Nature methods 12.9 (2015): 823-826. PubMed PMID: 26167643.
Additional Resources on the Addgene Blog
- Learn how to Choose the Best Cas9 Variant for Your Next Experiment
- Find out More about Genome-wide Screens with CRISPR/Cas9
- Learn How CRISPR Has Been Used with AAV
Resources on Addgene.org