Established in the mid 1970's, New England Biolabs, Inc. (NEB) is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development. New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and seven subsidiaries located in Canada, China, France, Germany, Japan, Singapore and the UK. For more information about New England Biolabs visit neb.
Do you need to test the effects of mutating a gene in your system? Then, CRISPR genome editing 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, and it can be hard to know which one to choose.
There are several factors to consider when deciding how to introduce CRISPR-Cas9 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 Explained!
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 a popular choice, but other retroviruses (such as MSCV) may be used depending on the cell type.
As with 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 you plan to use it frequently your project or in your lab. However, 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. If cost isn’t an issue you can even pay someone to make them for you, saving you even more time! Then, introduce them into your cells by electroporation or microinjection. You’ll have transient expression of Cas9 and the gRNA, which might be preferable, although you might lose some efficiency. Another drawback of this approach is that you won’t have a way to select for the cells that express both Cas9 and your gRNA. Again, this may or may not be a problem for your system.
Cas9-gRNA Ribonucleoprotein Complexes
In vitro transcription of Cas9 protein and gRNA ribonucleoprotein complexes bypasses the need for 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. This is done 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. Bear in mind that as with the mRNA/gRNA method, you won’t have any marker or selection available if you use Cass9-gRNA ribonucleoprotein complexes.
Overall, there are a handful of important considerations when choosing the system you’ll use to introduce CRISPR cas9 gene editing 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 are using. If not, you should be able to find some literature on the subject. It’s also worth doing a quick online search for CRISPR and your cell type of interest to compare the efficiency of different methods reported by different labs. CRISPR genome editing is a powerful technique, but you don’t need to reinvent the wheel when it comes to targeting your cells!
How do you get CRISPR components into your cells? Share your experiences with us by writing in the comments section!
Originally published in March 2017. Updated and republished in April 2017.Image Credit: KDS444