| Feature Articles |
April 24, 2017
The Most Interesting Thing about CRISPR/Cas9 Is What It Can Accomplish in the Hands of Gifted Researchers
The tooth-to-tail ratio, or the balance of frontline troops to support echelons, can determine success or failure in any complicated operation. Originally a military notion, the tooth-to-tail ratio also applies to commercial and scientific endeavors, such as the routinization of genome editing. Ideally, genome-editing technologies such as the CRISPR/Cas9 system should become so routine that they fade into the background.
Although CRISPR/Cas9 is a fascinating technology, the most interesting thing about this cutting and pasting machine, this RNA-guided endonuclease, is what it can accomplish in the hands of gifted researchers. Eventually, CRISPR/Cas9 will become just another tool, and everyone will go about their lives without having to hear about how “hot” it is. Before that happens, however, the other end of the CRISPR/Cas9 spear will need more attention and development. That is, CRISPR/Cas9 will need better supply, upkeep, and logistics.
Both parts of the CRISPR/Cas9 spear—the pointy research bit and the large support structure—were discussed at the CRISPR Precision Genome Editing Conference. This event, which recently took place in Boston, highlighted some exciting applications. What’s more, it included presentations that brought to mind the famous dictum, “Amateurs talk about tactics, but professionals talk about logistics.”
One application benefiting from CRISPR/Cas9 technology is xenotransplantation, or cross-species transplantation. It offers the prospect of an unlimited supply of organs and cells, and it could resolve the critical shortage of human tissues.
For ethical and compatibility reasons, xenotransplantation shifted away from nonhuman primates as a potential source of donor tissues. Instead, the discipline began to focus on porcine organs. Nonetheless, in 1997, pig-to-human transplants were banned worldwide due to concerns about the transmission to humans of porcine endogenous retroviruses (PERVs), which are integrated into the genome of all pigs.
According to George Church, Ph.D., professor of genetics, Harvard Medical School, work was undertaken in his laboratory on PK15 porcine kidney epithelial cells to determine if PERVs could be eradicated. It was crucial to avoid disrupting the envelope gene and the terminal regulatory elements, as both of these could be important during normal pig fetal growth. In addition, a highly conserved target in the viral polymerase gene was desired for the guide RNA (gRNA) to bind.
First, the copy number of PK15 PERV was determined to be 62. Then, when CRISPR/Cas9 was used along with two gRNAs, one which did the bulk of the work, all 62 copies of the PERV pol gene were disrupted, demonstrating the possibility that PERVs could be inactivated for potential clinical pig-to-human xenotransplantation. The repeats were well separated, and not clustered, which could have meant higher toxicity.
After two weeks of cell culture, about 8% of clones were 100% altered, and no rearrangements were found. Although a few off-target effects and point mutations were expected, they were deemed unlikely to have an impact on pig fetal development. As with conventional breeding, PERV-free clones were empirically selected as they were the healthiest.
In addition to disrupting dozens of endogenous viral elements, Dr. Church’s group altered dozens of genes involved in immune and blood-clotting functions to increase human compatibility. Some of the changes were so extensive that more powerful DNA recombination tools, and not CRISPR, were utilized.
This work may benefit eGenesis, a Cambridge biotech focused on leveraging CRISPR technology to deliver safe and effective human transplantable cells, tissues, and organs. eGenesis was cofounded by Dr. Church and Luhan Yang, Ph.D., in early 2015 and is based on their research.
Ex Vivo Indications
Another emerging company working on CRISPR clinical applications is CRISPR Therapeutics. This company’s initial emphasis is on ex vivo indications. Ex vivo indications have the benefit of a facile delivery approach, such as electroporation, and the ability to characterize the edits before administering treated cells to the patient. Plus, measuring biomarkers to understand phenotypic effects in a relatively short timeframe after therapy administration is straightforward.
The company’s lead indication, a compound to treat inherited single-gene hemoglobinopathies (such as sickle cell and beta thalassemia), relies on gene disruption to upregulate fetal hemoglobin. This approach could be curative. A large number of studies show that patients who have sickle-cell or beta-thalassemia traits are asymptomatic when they have upregulated fetal hemoglobin.
“We can achieve gene disruption today using CRISPR/Cas9 with relatively high efficiency, more than 80–90%,” asserted Sam Kulkarni, Ph.D., chief business officer, CRISPR Therapeutics. “Gene correction approaches are being improved continuously, but the efficiency of correction is still in the 50–60% range for hematopoietic stem cells.”
The key challenges to overcome include delivery, pharmacology, and manufacturing. CRISPR/Cas9 is a multicomponent system and needs to be delivered to the target organs or tissues for in vivo applications. Previous work on small interfering RNA (siRNA) and other therapeutic modalities may prove beneficial.
Careful analysis is required to characterize the type of edits and the fraction of cells edited. This pharmacology hurdle is easier to clear ex vivo than in vivo. Finally, manufacturing involves multiple components and also complex cell manufacturing. Collective efforts of private and academic laboratories are rapidly surmounting these issues.
Recent advances that result in high levels of homology-directed repair are facilitating efforts to expand the addressable indications with CRISPR. Some approaches attempt to impact the level of cycling of the cells; others utilize modifications of the donor template and guides; and yet others are working on optimization of the process of CRISPR/Cas9 directed repair.
Arrayed CRISPR Libraries
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MilliporeSigma participated in the development of the Sanger Arrayed Whole Genome Lentiviral CRISPR Libraries. According to the company, these are the first commercially available off-the-shelf arrayed lentiviral CRISPR gene knockout libraries for screening human and mouse genomes. The genome-wide loss-of-function screens won an R&D 100 Award in 2016.
Tools play an important role in making the prospect of high-throughput knockout screening a reality. Such tools have been pioneered by MilliporeSigma, which has launched various CRISPR products. The first such product consisted of simple constructs/plasmids that could accommodate targeting elements and yield custom clones. Soon after developing this product, MilliporeSigma realized that a large collection of clones would be of great utility.
A previous collaboration between MilliporeSigma and the Broad Institute had resulted in a short hairpin RNA (shRNA) library, and the goal was to duplicate that model to develop a product that would work for the majority of researchers.
Another collaboration, this one between MilliporeSigma and the Wellcome Trust Sanger Institute, had a similar vision, and after two years of work, it generated its first complete arrayed whole-genome CRISPR screening libraries. The off-the-shelf libraries offer substantial cost savings and facilitate standardization of CRISPR screening. Although pooled libraries have been available for a while, the arrayed libraries provide one clone for one gene in one well, reducing ambiguity about the target at screen completion.
The human and mouse libraries were designed to knock out virtually every protein coding gene in their respective genomes, and each library contains two unique and highly specific gRNAs for every gene target. The second clone is used to verify that the first is not an artifact or a false positive.
The human library contains approximately 34,000 clones targeting 17,000 genes, and the mouse library contains approximately 40,000 clones targeting 20,000 genes. Bacterial glycerol stock, plasmid DNA, and lentiviral formats are available.
“The beauty of the Sanger designs is that they will hit that gene and that gene only,” commented Shawn Shafer, Ph.D., director, advanced genomics, MilliporeSigma. “Anything that did not meet our stringent design strategy did not make it into the library.
“Some genes are tiny or highly repetitive, and so these genes were not suitable targets. Now you can screen for one gene, a couple, or the whole genome.”
The arrayed library will continue to evolve. Dialing a gene up or down is looking plausible, and the libraries may be developed for use in gene activation and repression.