Do you need to test the effects of mutating a gene in your system? Then, CRISPR is the way to go. Your first step is to decide on good target sequences. Then, you have to get the two components of CRISPR: the Cas9 nuclease and “guide” RNA (gRNA). Even though you’ve read up on the technique online, there are many ways to get these components into cells—which one should you choose? There are several factors to consider when deciding how to introduce CRISPR-Cas into your cells. Here’s a list of some possible methods, and what you should consider when deciding between them:
Plasmid/Expression Vectors for CRISPR
You will need one plasmid containing a sequence encoding Cas9 and the same or another plasmid for your chosen gRNA sequence. Addgene and others have made such sequences widely available, including versions with a variety of promoters—both constitutive and inducible. You will probably have to clone your gRNA of interest into a vector, unless you’re lucky enough that someone else has already done so. Addgene lists and distributes many experimentally validated gRNAs.
These vectors provide a lot of flexibility—do you want Cas9 stably expressed or would transient expression be better? Do you want to express Cas9 and your gRNA in the same vector or different vectors? Note, the ability to use separate vectors for Cas9 and your gRNA allows you to flexibly combine Cas9 with different gRNAs. What promoters work well in your cell type? Would you like to use a selection marker or perhaps simply a tag to mark those cells that have been successfully transfected? Keep in mind that this does not mean they’ve been successfully edited! While using expression vectors provides a lot of flexibility, the efficiency is highly dependent on your cells of interest.
Viral Vectors for CRISPR
Another option is to introduce the DNA encoding Cas9 and your gRNA into cells using viral vectors. Lentivirus is frequently used, but other retroviruses (such as MSCV) may be used depending on the cell type. Like using plasmids, this method will generally require you to clone your gRNA sequence into a vector. And the efficiency will depend in part on the transduction efficiency you can achieve in the cells you chose. You have the option of introducing a selection marker or other tag to identify transduced cells and the ability to use separate vectors for Cas9 and your gRNA. As with stable transfection, viral integration may have a negative impact on your cells, so keep this in mind if you choose one of these methods.
Cloning a vector might be worthwhile if it will be a commonly used resource for your project, lab, or others. But if you want to test the effect of a specific alteration, one of the following methods might work better.
Cas9 mRNA and gRNA
If you don’t want to spend time cloning your gRNA sequences into vectors with the appropriate promoter, or your cells are hard to transduce and transfect, then you can transcribe in vitro Cas9 mRNA and your gRNA of interest (or pay someone to make them for you if cost isn’t too much of an issue!). Then, introduce them into your cells by electroporation or microinjection. You’ll have transient expression of Cas9 and the gRNA, which might be preferable, you might lose some efficiency. There also isn’t a good way to select for or mark which cells are expressing both Cas9 and your gRNA. Again this may or may not be a problem for your system.
Cas9-gRNA Ribonucleoprotein Complexes
In vitro formation of Cas9 protein and gRNA ribonucleoprotein complexes requires no cloning and takes less time than waiting for translation of Cas9. To use this method, you in vitro transcribe your gRNA of interest and complex it with commercially produced Cas9 protein (simply by mixing the two components together and leaving them at room temperature for 15 minutes!). This complex is then introduced to your cells by electroporation or using cationic lipids. Like the mRNA/gRNA method, you won’t have any marker or selection available.
Overall, there are a handful of important considerations when choosing the system you’ll use to introduce CRISPR components into your cells. You’ll need to consider the time to obtain/make reagents, transient or stable expression, whether you need selection or a marker, efficiency (for your cell type!), and the effect on cell viability. You might already know what type of transfection or transduction method works well in the cells you need. If not you should be able to find some literature on the subject. It’s also worth a quick search for CRISPR and your cell type of interest to compare the efficiency of different methods that have been reported by different labs. CRISPR is a powerful technique, but you don’t need to reinvent the wheel when it comes to targeting your cells!
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