Cutting-Edge CRISPR Tools

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New products make gene editing as easy and effective as possible

Everyone who pays attention to science or technology knows that CRISPR is the hottest new method for customizing an organism’s genes. These clustered regularly interspaced short palindromic repeats come from part of a bacterium’s defense system, and CRISPR can edit DNA in specific spots. People know of this technology either through scientific articles, the patent battles, or the many mentions in the media and online—on September 2, 2017, a Google search for CRISPR returned more than seven million hits.

Despite calling this the hottest new method for gene editing, it’s not that new. In 1993, microbiologist Francisco Mojica of the University of Alicante, Spain, described these genetic sequences. But 20 years passed before Feng Zhang, the James and Patricia Poitras Professor in Neuroscience at MIT, reported using CRISPR to edit genes in cells from mice and humans.

Now, fewer than five years after Zhang’s report, scientists can purchase many tools and kits that make it easier to use CRISPR. The process involves a CRISPR-associated protein, like Cas9, and guide RNA, which determines where Cas cuts.

Reasons for ribonucleoprotein

Using CRISPR for gene editing, says Mark Behlke, chief scientific officer at IDT (Coralville, IA), “changed the whole game.” That game, though, keeps changing. The first uses of CRISPR in 2013 delivered the editing tools to an organism through plasmids, and many scientists still use this technique, but the same can be accomplished with a ribonucleoprotein (RNP).

To make it easier to use CRISPR with fewer off-target effects, IDT developed the Alt-R S.p. Cas9 Nuclease 3NLS. Using the recombinant Cas9 protein as an RNP, says Behlke, “the editing is all done in 48 hours, but a plasmid system can overexpress Cas9 protein for a week.” Use of the RNP system provides fast-on and fast-off editing, which is both efficient and more specific.

U2OS cell line with multiple fluorescent reporters generated through multiplexed CRISPR-Cas9-mediated knock-in, including use of the Edit-R HDR Donor Designer. (Image courtesy of Dharmacon.)

To further reduce off-target effects, scientists at IDT screened about 250,000 mutants of Cas9 for activity in gene editing. “From this, we came up with a single-point mutation that reduced off-target cutting, but retained nearly full activity at the target,” Behlke explains. “It works at 90% of the sites that the wild type does.” The degree of reduction in off-target effects depends on the site studied, but Behlke has seen it go down tenfold or more in some cases.

Beyond increased accuracy, the RNP method is easy. “The RNP complex can be delivered by lipofection in some cases,” Behlke notes. If that doesn’t work, electroporation works for most cell lines, including stem cells, Behlke points out. “We have customers using primary human T cells, and they can get the desired editing in 80% of the cells with one electroporation,” he says.

Success with single guide

At New England Biolabs (Ipswich, MA), scientists can buy CRISPR kits based on single-guide RNA (sgRNA). “The EnGen sgRNA Synthesis Kit, S. pyogenes, provides a simple and quick method for transcribing high yields of sgRNA in a single 30-minute reaction, using the supplied reagents and target-specific DNA oligos designed by the user,” says Breton Hornblower, product marketing manager for RNA, genome editing, and synthetic biology at New England Biolabs. “We have coupled the process of DNA template generation and RNA transcription in one simple reaction.”

The company developed this kit to bring CRISPR to every interested lab. “Our internal research required the testing of many different guide RNAs,” Hornblower explains. They could have created the guide RNA through in vitro transcription, but that requires making templates for every guide. Customers could have also ordered synthetic guide RNA, but that’s more expensive and takes time. “To get around this,” Hornblower explains, “we developed a product that would only require a target specific 55-mer single-strand DNA [ssDNA] oligo, which is inexpensive with no real lead time.”

The kit comes in handy when scientists lack a guide template. “It allows them to pick a target and then order a ssDNA oligo containing that target and then create microgram quantities of that guide very quickly,” Hornblower says. “It is also very useful when a customer has many different guides they want to try, and they can order one ssDNA oligo for each target to be used with the kit.”

Precise genomic alterations

At Dharmacon, Horizon Discovery (Lafayette, CO), one of the latest CRISPR products is the Edit-R HDR Plasmid Donor Kit. As described by Anja Smith, director of research and development at Dharmacon, Horizon Discovery, the kit is used “for insertion of fluorescence reporters—mKate2 or EGFP—or a customer-defined DNA sequence into almost any genomic location.” It uses the homology directed repair, or HDR, pathway plus CRISPR. “The HDR Plasmid Donor Kits are complemented by free online design tools for optimal guide RNA design, as well as design of custom kit components,” Smith explains. These design tools can be applied to more than 35 species, including fish, insects, plants, and reptiles.

When asked why Dharmacon developed this product, Smith says, “Using CRISPR-based approaches to modify a cell’s genome is of high interest across many fields of study, including cancer genomics, infectious disease, and host-pathogen relationships.” She adds, “The assembly of reagents required for doing these experiments can be complicated and time-consuming.” Combining the company’s CRISPR Design tool and custom guide RNAs with the HDR Donor Designer and Edit-R HDR Plasmid Donor Kits, says Smith, “significantly reduces the hands-on design time and effort required to successfully insert a mKate2, EGFP, or custom sequence into cells.”

These tools can be applied in many ways, such as inserting fluorescent markers in a specific spot in a genome. “With these tools, researchers can use fluorescence to characterize the localization of their target proteins, characterize binding partners, and quantify changes in transcription or translation due to experimental conditions,” Smith explains. “Using a custom insert, researchers may insert or modify entire exons to model diseases for better understanding of etiology and accelerate the development of treatments.”

Analyzing the edits

Although today’s tools make CRISPR easier and more efficient than ever, it’s not perfect. Boris Fehse, a biomedical scientist at Germany’s University Medical Centre, Hamburg-Eppendorf (UKE), and his colleagues noted that “detection and quantification of gene-knockout frequencies … has remained a technical challenge.”¹

Consequently, researchers need to check the results. “Scientists should run validation of gene editing after CRISPR,” says Carolyn Reifsnyder, senior marketing manager in the digital biology group at Bio-Rad Laboratories (Hercules, CA). This can be done in various ways, including quantitative or droplet digital PCR—qPCR and ddPCR, respectively.

If high sensitivity is not needed, qPCR provides a quick check that works as long as 20% or more of the cells are edited correctly. “If you want to know if less than 1% of the cells got edited, then ddPCR is where you should be,” Reifsnyder explains.

Fehse and his colleagues used ddPCR for “concurrent quantification of edited and wild-type alleles in a given sample.” They concluded: “We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings.”

Bio-Rad can supply genome edit–detection assays for any target. “The assays drop into our QX200 Droplet Digital PCR system and users can detect editing events present at frequencies of less than 0.5%, from as little as 5 nanograms of total genomic DNA,” Reifsnyder explains.

With tools like the ones described here, any lab can put CRISPR to work. Nonetheless, scientists must also make sure to validate the results before moving forward. Only then can they discover everything that today’s most powerful gene-editing tool can do.

Reference

1. Mock, U.; Hauber, I. et al. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nature Protocols 2016, 11, 598–615.

Mike May is a freelance writer and editor living in Texas. He can be reached at mike@techtypercom.