Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
In the world of genome editing, CRISPR/Cas9 stands supreme. In providing researchers with a simple method for directing a nuclease, transcriptional regulator or fluorescent protein to any genomic address they desire, the method has transformed research focused on disease etiology, gene regulation, drug development and more.
First, though, researchers must direct the necessary molecular machinery—the Cas9 protein and guide RNA (gRNA)—to the cells of interest. As it turns out, they have several options.
Special delivery
The CRISPR/Cas9 system represents one of a small number of examples in which researchers have the freedom to transfect DNA expression vectors, messenger and gRNAs or protein. According to Xavier de Mollerat du Jeu, director of research and development for the transfection group at Thermo Fisher Scientific, the decision comes down to how much protein researchers need in the cell, and for how long.
The CRISPR/Cas9 system represents one of a small number of examples in which researchers have the freedom to transfect DNA expression vectors, messenger and gRNAs or protein.
DNA expression vectors, he explains, tend to produce high-level expression of protein and RNA over several days; but there’s a delay, as the genes must be transcribed and (in the case of Cas9) translated. With Cas9 mRNA and gRNAs, editing occurs more rapidly, as no transcription is required, but it drops off quickly as the transcripts get degraded. Protein, of course, is fastest of all, as neither transcription nor translation is necessary. And as with RNA, protein turns over rapidly.
When it comes to Cas9, says Veronika Kortisova, global product manager at Bio-Rad Laboratories, rapid turnover is actually a plus, as it yields fewer off-target events than does long-term expression. “The hypothesis is that when the system goes in, it does the edit and leaves the cell—it’s less toxic. With plasmids, the persistent protein expression may sometimes lead to negative effects.”
But there are also downsides to using Cas9, says Laura Juckem, research and development group leader at Mirus Bio. Most significantly, protein and mRNA require extra work to make and purify in the lab, and they cost more than plasmid DNA. Researchers can, however, purchase purified Cas9 protein and/or mRNA from several vendors, including GE Healthcare Dharmacon, MilliporeSigma, Miltenyi Biotec, New England Biolabs and Thermo Fisher Scientific.
Delivery tools
Researchers generally have two options for nucleic acid delivery: transfection reagents and electroporation.
Reagents for transfection use either lipids, polymers or some combination thereof. Some reagents are designed to be specific to a particular type of target, such as Bio-Rad’s siLentFect™ for siRNA, Thermo Fisher Scientific’s Lipofectamine™ MessengerMAX for mRNA and Promega’s DNA-optimized FuGENE reagent; others, like Bio-Rad’s TransFectin reagent, Mirus Bio’s TransIT-X2 and Thermo’s Lipofectamine 3000, support a broader range of applications.
Few reagents, however, are specifically capable of delivering protein. Two exceptions are Clontech’s Xfect Protein Transfection Reagent and ActiveMotif’s Chariot™ Protein Delivery Reagent. According to Cornelia Hampe, product manager at Takara Bio Europe (Clontech Laboratories’ sister company in Europe), the Xfect reagent is a cell-penetrating peptide capable of delivering proteins as large as 400 kDa; to use it, simply mix the peptide with the protein of interest and add the resulting complex to the cells. “It coats the protein to make it pass [through] the membrane,” she says.
Electroporation, on the other hand, is relatively agnostic with respect to the molecules being delivered. The method punches holes in the cell membrane; if a molecule can pass through the breach, it can enter the cell—and that includes Cas9-gRNA complexes. “When it comes to delivering CRISPR/Cas9 tools as plasmid DNA, mRNA or ribonucleoprotein, Nucleofection works very efficiently with any of these,” says Andrea Toell, senior product manager at Lonza, whose Nucleofector platform enables electroporation of a wide range of cell types, including hard-to-transfect primary cells. And, she adds, “Since you can use the same conditions for any substrate, you can even efficiently co-transfect different types.”
Toell says Lonza offers “a broad range of ready-to-use protocols, so customers can start their scientific experiments right away rather than having to optimize conditions.” Similarly, Bio-Rad has created a searchable database of electroporation protocols organized by cell type for its GenePulser Xcell, and Thermo Scientific has done likewise for its Neon® system.
Recently, CRISPR-Cas9-specific delivery tools have come on the market. Thermo Scientific’s Lipofectamine CRISPRMAX™, for instance, is a new Lipofectamine formulation specifically designed to deliver the CRISPR-Cas9 complex to cells. By pairing GeneArt Platinum Cas9 Nuclease and Lipofectamine CRISPRMAX Cas9 Transfection Reagent, you can significantly simplify the cell engineering workflow and achieve cleavage efficiencies up to 85% in many cell lines.
Another option is Clontech’s Guide-it™ CRISPR/Cas9 Gesicle Production System. A gesicle, Hampe explains, is a glycoprotein-studded nanovesicle created in a producer cell line. Researchers clone their gRNA into a dedicated expression vector and deliver that plasmid, plus plasmids expressing viral glycoproteins, Cas9 and a membrane-bound red fluorescent protein, to 293T cells. After the system is induced, Cas9/gRNA-filled vesicles bud from the cells into the supernatant, from which they can be harvested, concentrated and added to target cells.
Application considerations
There are other occasions for which delivery of DNA, RNA and protein are all viable options. One is the generation of induced pluripotent stem cells (iPSCs). Viral and plasmid-based reprogramming kits are available, as are mRNA and self-replicating RNA systems, including products from MilliporeSigma and Stemgent.
As for protein, in 2009 Sheng Ding of The Scripps Research Institute in La Jolla, Calif., and colleagues fused a poly-arginine peptide to the four Yamanaka reprogramming factors, Oct4, Sox2, Klf4 and c-Myc, demonstrating the feasibility of protein-mediated reprogramming [1].
According to Frank Hsiung, a staff scientist at Bio-Rad, the decision of which macromolecule to deliver depends in part on application. “The purpose of transfecting something is so the newly introduced elements can regulate gene expression,” he says. “It all depends on how [researchers] want to play with the gene of interest.”
If the goal is gene silencing, for instance, it may be enough to deliver an siRNA to the cytoplasm. If rapid activity is key (say, using protein to influence cell behavior at specific time points), then protein delivery makes more sense.
Sometimes, though, the decision is based not on experimental need, but on the target cells themselves. Some cells are simply difficult to transfect—at least with plasmid DNA. But what’s true of plasmid is not necessarily true for other molecules. “There’s a common belief that if a cell is hard to transfect, it’s hard to transfect no matter what you use,” de Mollerat du Jeu explains. But some cells that are refractory to DNA delivery tolerate mRNA and protein just fine. “And the reason is because DNA needs to go into the nucleus in a cell,” but mRNA and protein do not. (Among other things, nuclear delivery often requires that cells be actively dividing, as it is the breakdown of the nuclear pore during division that allows DNA to enter the nucleus; but mRNA and protein only need to reach the cytoplasm.)
“There’s a holy grail of transfection,” Kortisova says. It is “finding a tool that delivers high viability, high efficiency and is universal.” At the moment, no such technology exists, she concedes. But researchers are getting closer all the time. In the meantime, researchers interested in the CRISPR/Cas9 system have plenty of options.
Reference
[1] Zhou, H, et al., “Generation of induced pluripotent stem cells using recombinant proteins,” Cell Stem Cell, 4:381-4, 2009. [PMID: 19398399]
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