Supplement: CRISPR Shakes Up Tools and Tech

Innovators Are Developing More Precise DNA-Cutting and Single-Cell-Seeding Platforms

CRISPR, the famously user-friendly gene snipping tool, has shaken up not only the world of clinical therapeutics but also the tools and technology used in research. Speakers at the recent Precision CRISPR Congress 2018 highlighted an array of the latest CRISPR-centric tech emerging from their companies or labs.

Unexpected Tech

CRISPR-Cas12a is like a cousin to the widely used CRISPR-Cas9 system. It, too, has a programmable guide RNA (gRNA) to direct the system to the target DNA sequence so it can create double-stranded DNA breaks. However, unlike Cas9, Cas12a achieves this with only one catalytic domain, not two. Because of this mechanistic difference, co-inventor of CRISPR-Cas9 technology, Jennifer Doudna, Ph.D., and her lab at the University of California, Berkeley, as well as other colleagues, sought to understand why.

“While we were trying to investigate this mechanism, we found this unexpected property,” said Janice Chen, a Ph.D. candidate in Dr. Doudna’s lab. Upon binding to the target DNA sequence, Cas12a would indiscriminately shred any single-stranded DNA. “What this suggested was that we could actually use this as a way of having a targeted detection platform.”

At the meeting, Chen debuted the team’s diagnostic platform, coined DETECTR (DNA endonuclease targeted CRISPR trans-reporter); the platform was also published in Science (Figure 1).1

Figure 1. The DETECTR
platform developed at UC Berkeley uses CRISPR-Cas12a to analyze cells, blood, saliva, urine, and stool, enabling the detection of genetic mutations, cancer, and antibiotic resistance, as well as the diagnosis of bacterial and viral infections. In the DETECTR system, Cas12a finds and binds target DNA that has been amplified by recombinant olymerase. After Cas12a cuts a targeted double-stranded DNA sequence, it indiscriminately
cuts single-stranded DNA, including DNA attached to a fluorescent marker (gold star), which tells researchers that Cas12a has found its target.

The platform works by programming Cas12a and its gRNA to a specific target sequence. To enable detection, the system also requires a reporter molecule, which has a fluorescent molecule attached to one end and a quencher to the other. Before Cas12a reaches the target DNA sequence, the single-stranded DNA reporter molecule is intact and no fluorescent signal appears. But when Cas12a binds to its target DNA sequence, it also cleaves the reporter molecule, causing the fluorophore to break apart from the quencher. A fluorescent signal then appears.

The diagnostic platform has the potential to detect an array of diseases, including bacterial and viral infections, cancer, as well as genomic characteristics. The platform has already been successful in experiments for detecting specific human papilloma virus sequences. “[It finds] anything that has any basis in nucleic acids,” summarized Chen.

Driverless Cell Seeding

Figure 2. Solentim has introduced a single-cell printer called VIPS™, which refers to verified in situ plate seeding. VIPS can isolate and dispense individual cells into microtiter plates and then immediately image the wells in the microtiter plates to confirm that individual wells contain single cells. According to Solentim, VIPS should facilitate the cell engineering projects in which CRISPR systems modify cancer or tissue cell lines.

Ian Taylor, Ph.D., sales and marketing manager at Solentim, presented a new technology for single-cell seeding—VIPS™ (verified in situ plate seeding), a fully integrated system for automated, single-cell cloning (Figure 2). CRISPR gene editing groups can use the VIPS system to isolate single gene-editing clones to grow them into single colonies that can then be screened for the desired mutation.

Overall, the VIPS system performs single-cell seeding by releasing a cell-containing droplet into the bottom of a dry well. The system acquires an image to validate that it did indeed deposit a single cell into the well. Then the system fills that well with media and snaps an image of the whole well. The single cell eventually grows into a single colony. The system also has a few extra bells and whistles, like fluorescent labels and an electronic report feature that documents the process at multiple time points.

Dr. Taylor explained the major differences between the VIPS system and current single-cell seeding approaches, namely limiting dilutions and fluorescence-activated cell sorting (FACS).

Figure 3. According to Solentim, the VIPS platform takes about 10 minutes to carry out a single-cell printing procedure that completely seeds a full 96-well plate. Results are displayed in the form of a plate map indicating empty wells, wells with single cells, and wells with multiple cells. High-speed, whole-well imaging can be carried out by VIPS for validation (using fluorescence) experiments.

With limiting dilution, he said, 80% or 90% of the wells in the plate do not show growth, making it an inefficient process. Dr. Taylor added that even when a single colony does grow in a well, people may mistakenly conclude that the single colony came from a single cell: “There’s lots of reasons subsequently why that is not the case.”

With the VIPS system, about 90% of the plate is successfully seeded with single cells (Figure 3).

As for the FACS approach, Dr. Taylor noted that it is “not really designed” for single-cell seeding. Despite achieving high purity, FACS dispenses cells in a way that actually destroys a lot of the cells in the process.

“You’ll find customers using the FACS approach get very, very low recovery and growth of cells in wells,” he said. “The VIPS system is a gentle method and ensures high efficiency of single cells in wells. Most of those single cells will grow into colonies, so the cloning efficiency will also be high.”

Custom Libraries—and More

Since CRISPR hit the scene in 2012, many companies have begun offering their own CRISPR libraries to scientists who are conducting genetic screening experiments.

Leon Song, Ph.D., director at GenScript, presented the latest GenScript tools, one of which is called CRISPR Libraries. “Currently, we have three kinds of libraries,” said Dr. Song. The libraries include a genome-wide loss-of-function library, which is a Broad Institute–designed GecKOV2 library of human and mouse genes; a genome-wide gain-of-function library, which is a synergistic activation mediator (SAM) library of human and mouse genes; and pathway-based gRNA libraries for targeted pathway screening.

He also presented GenScript’s CRISPR ribonucleoprotein (RNP) system, which is part of the company’s GenCRISPR™ Cell Line service. The CRISPR RNP system consists of a CRISPR RNA that complements the target DNA, trans-activating RNA that helps guide the Cas9 protein, and a Cas9 endonuclease. According to Dr. Song, “Our new system provides high efficiency, low toxicity, and a shorter turnaround time.”

Namritha Ravinder, Ph.D., senior manager of R&D at Thermo Fisher Scientific, discussed several new tools, one being the Invitrogen LentiArray CRISPR Libraries, a platform for identifying novel targets in biological pathways and disease development. The LentiArray CRISPR libraries earned the number five spot in The Scientist top 10 innovations for 2016.

As mentioned in Dr. Ravinder’s talk, the LentiArray CRISPR Libraries were field-tested by Simone Treiger Sredni, M.D., Ph.D., associate professor of pediatric neurosurgery at Northwestern University Feinberg School of Medicine, and her team. The study was published in Pediatric Blood & Cancer in 2017.2

Dr. Sredni and colleagues were aiming to identify a therapeutic target for a lethal pediatric brain tumor, malignant rhabdoid tumor, so the team used the LentiArray CRISPR Libraries to screen 160 kinases in tumor cells to determine which kinases control cell proliferation and growth. The team determined that Polo-like kinase 4 (PLK4) has a role in cellular proliferation, identifying the enzyme as a potential therapeutic target. Next, the team tested a PLK4 inhibitor (CFI-400945) in multiple rhabdoid tumor cell lines. The outcome was encouraging: tumor shrinkage.

“The LentiArray CRISPR libraries have allowed us to make significant inroads in our research on pediatric cancer, discovering a potentially novel strategy for the treatment of deadly brain tumors,” said Dr. Sredni.3

Dr. Ravinder also highlighted the Invitrogen Cas9 RNP system, including TrueCut Cas9 V2 protein and TrueGuide gRNA. She explained that her team was able to use the Cas9 RNP system in a series of genome editing experiments with human primary T cells where they showed over 90% efficiency.

“When we say over 90% efficiency, we’re showing functional knockout of a particular gene, not just cutting the genome,” stated Dr. Ravinder. “Previously, it was really hard to get such high efficiencies when attempting gene modulations for knockouts in primary cells. It wasn’t just the lack of tools. It was also the lack of a good protocol.”

“If editing efficiency is high,” she added, “when people take those T cells and then put them into a mouse for their work, they have a better chance of seeing the particular phenotype that they’re interested in.”


Christina Bennett is a Freelance Writer for GEN.