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.
Several months ago, the news broke that the first clinical trial to use CRISPR genome editing in human patients had “passed a key safety review.”
As reported in Science, Carl June, of the University of Pennsylvania Perelman School of Medicine, and colleagues got the green light June 21 from the U.S. National Institutes of Health’s Recombinant DNA Advisory Committee to move ahead with a proposed trial using CRISPR to modify two genes in the T cells of patients with certain cancers, which would then be reinfused back into the patients to attack the diseased cells.
The researchers have some hurdles to overcome before the trial can begin. And how the trial will fare, of course, is anybody’s guess. But the fact that CRISPR technology has achieved this milestone, just four years after its discovery, is remarkable. Indeed, as outlined in one 2015 review, researchers are exploiting CRISPR technology in all aspects of cancer research, from disease modeling to drug screening to therapeutics [1].
Jon Moore, chief scientific officer at Horizon Discovery, a company that offers both custom modified cells and CRISPR-based services, sees big roles for genome editing in drug target identification and validation, as well as in the generation of direct cellular therapeutics. For instance, Horizon has run proof-of-concept pooled library screens, looking for genes that interact with p53, a tumor suppressor commonly (but not always) mutated in cancer. That screen identified MDM2, a different tumor suppressor, as another potential drug target, Moore says. “In cancer cells that retain wild-type p53 activity, they are very sensitive to knockout of MDM2.”
For those who would perform such studies in their own labs, there exists a robust and growing set of tools.
gRNA libraries
One of the most useful tools for those hoping to leverage CRISPR in pursuit of anticancer therapeutics is a guide RNA library.
One of the most useful tools for those hoping to leverage CRISPR in pursuit of anticancer therapeutics is a guide RNA library .
These libraries typically come in one of two forms. An arrayed library is distributed into the wells of one or more microtiter plates, with each well containing free gRNAs (or a gRNA-expression vector) targeting a single gene. Screening conditions depend upon the specific application, but such libraries can be used, for instance, to identify genes that enhance tumor drug resistance.
Thermo Fisher Scientific’s Invitrogen™ LentiArray™ libraries are arrayed lentiviral libraries with four gRNAs per gene, says Jon Chesnut, the company’s research and development head for synthetic biology and genome editing. The company is working its way through the entire human genome, Chesnut adds; however, at the moment, only specific subsets, including the human kinome and GPCRs, are available.
MilliporeSigma’s Sanger Human Whole Genome Arrayed Lentiviral CRISPR library, built in conjunction with the Wellcome Trust Sanger Institute, comprises some 40,000 gRNAs, with two gRNAs for every human and mouse gene.
A pooled library is one in which all the gRNAs to be screened are mixed in a single tube. This mixture is then transduced into cells, each of which should pick up one expression vector. By then subjecting the cells to some positive or negative selection, such as drug treatment, and deep-sequencing the survivors, researchers can determine which gRNAs (and thus target genes) were most effective.
“It’s significantly more difficult to get to the answer,” concedes Chesnut. “But in labs that aren’t set up to do high-throughput screening, the pooled-library approach is a great alternative.”
Pooled libraries are available from GE Life Sciences/Dharmacon, MilliporeSigma and Agilent Technologies, among others. In February, Agilent launched an early-access program for a library based on the Broad Institute’s GeCKO v2 pooled lentiviral library for human and mouse, comprising a total of 123,411 guides for human, according to Caroline Tsou, global marketing director for molecular and synthetic biology at Agilent Technologies. (The company also offers a custom library, which can deliver up to 200,000 guides of any design.)
More recently, an updated version of the GeCKO library was released. It is available through Addgene and MilliporeSigma [2].
MilliporeSigma also has a gRNA library in development that’s specifically intended for use with its dCas9-p300 activator construct, says Patrick Sullivan, head of innovation research and development for gene editing. In this case, rather than knocking out or repairing a gene, the library is designed to upregulate the expression of targeted genes. Such a library, Sullivan says, could be used to identify genes whose activation serves to make a potential therapeutic more effective, or to identify new druggable targets. The library is expected to launch in 2017, he says.
CRISPR delivery
Cornelia Hampe, scientific support specialist and product manager at Takara Bio Europe, offers two pieces of advice for those designing CRISPR-based experiments. First, design multiple guides per target—as many as four or five—to counter the likelihood of off-target effects. Design those guides using multiple tools, she adds, and test them in vitro prior to use in cells (ie., using Takara's Complete sgRNA Screening System).
Her other bit of advice is to control the strength and duration of Cas9 expression, especially when developing therapeutics.
“Most often, if it’s very strong, stable expression, this can lead to off-target effects,” she says.
“With therapeutics, one wants to ensure the off-target effects are as low as possible.”
How does one do that? Though researchers have multiple options for delivery of Cas9 and gRNAs, including DNA plasmid-based expression, viral vectors, in vitro transcription systems and purified mRNAs, and even intact ribonucleoprotein complexes, most experts say that when it comes to CRISPR-Cas, the smart money is on transient delivery.
“The most popular and effective [Cas9 format] at the moment is Cas9 as purified protein or as purified RNA,” says Chesnut. In the former case, researchers complex purified Cas9 with in vitro transcribed RNAs and deliver that either via electroporation or using a specialized reagent such as Thermo’s Lipofectamine™ CRISPRMAX™.
A related option is Takara’s “gesicle” (glycoprotein vesicle) technology, launched in January. Gesicles, explains Hampe, are glycoprotein-studded nanovesicles. Loaded with Cas9/sgRNA ribonucleoprotein complexes within a dedicated packaging cell line, the particles have a broad cellular tropism and can deliver to both dividing and nondividing cells. They possess a red, fluorescent protein marker that lights up transduced cells. And, like other RNP-based methods, they produce a rapid burst of editing followed by rapid clearance.
“The delivery works in a broad variety of cells,” Hampe says. “Especially in hard-to-transfect cells, like Jurkat and retinal pigment epithelium, it was much better compared to plasmid.”
Organoid cultures
Another useful tool for CRISPR-based cancer research is a 3D cell-culture approach called organoid culture. As described in one recent review, an organoid is “an in vitro 3D cellular cluster derived exclusively from primary tissue, ESCs or iPSCs, capable of self-renewal and self-organization, and exhibiting similar organ functionality as the tissue of origin” [3].
Organoids, the review asserts, “represent an important bridge between traditional 2D cultures and in vivo mouse/human models, as they are more physiologically relevant than monolayer culture models and are far more amenable to manipulation of niche components, signaling pathways and genome editing than in vivo models.” As Alex Sim, president of AMS Biotechnologies, a company that sells reagents for organoid cell culture, puts it: “There’s no plastic in your body.”
With organoids and CRISPR, Sim says, researchers could introduce mutations into normal cells (or correct mutations in cancerous cells), then monitor the cells’ proliferative capacity or response to drugs, as well as their transcriptomics and/or protein expression profiles.
In one recent example, researchers led by Hans Clevers at the Hubrecht Institute in Utrecht, the Netherlands, used CRISPR gene editing to introduce four mutations commonly associated with colorectal cancer into wild-type human intestinal and colon epithelial cell organoids [4]. The resulting cultures exhibited chromosomal instability, a reduced requirement for growth factors and formed tumors when injected into immunodeficient mice, suggesting they represent a realistic model of colon carcinoma.
Any advances arising from such models, of course, will take years to work their way through development and into the clinic. But given the pace of CRISPR research to date, it’s a safe bet that new tools are on the way. And soon.
References
[1] Sánchez-Rivera, FJ, Jacks, T, “Applications of the CRISPR-Cas9 system in cancer biology,” Nature Reviews Cancer, 15:387-95, 2015. [PMID: 26040603]
[2] Sanjana, NE, Shalem, O, Zhang, F, “Improved vectors and genome-wide libraries for CRISPR screening,” Nature Methods, 11:783-4, 2014. [PMID: 25075903]
[3] Fatehullah, A, Tan, SH, Barker, N, “Organoids as an in vitro model of human development and disease,” Nature Cell Biology, 18:246-54, March 2016. [PMID: 26911908]
[4] Drost, J, et al., “Sequential cancer mutations in cultured human intestinal stem cells,” Nature, 521:43-7, 2015. [PMID: 25924068]
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