DNA Gains New Bases, Keeps Shapely Helix

Scientists have created a double helix out of six nucleotides,
 two more than what nature had devised.
[American Chemical Society]

Additions to DNA’s four-letter alphabet have been announced before, but the new letters don’t always fit comfortably into DNA’s double helix. Even slightly awkward letters can distort DNA’s overall shape and alter the molecule’s plasticity. Worse, a sequence of awkward letters that omits natural letters can amplify any structural problems, limiting new-letter-enriched synthetic DNA’s ability to interact with structural proteins and enzymes, jamming life’s molecular machinery.

A new pair of letters, however, seems to slide into DNA fairly easily. The new letters, or nucleotide bases, are called Z and P. They emulate structural and functional features of DNA’s natural nucleotide bases, C and G (cytosine and guanine) and A and T (adenine and thymine). Not only can they be interspersed with the natural nucleotide bases, they can be run adjacent to each other in a sequence, and the resulting DNA will still form a nice helix. What’s more,

the resulting DNA even shows the ability to evolve.

Z stands for 6-amino-5-nitro-2(1H)-pyridone. P stands for 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one. Z and P were introduced in a pair of papers prepared by Millie M. Georgiadis, Steven A. Benner, and colleagues from Indiana and Florida, and published in the Journal of the American Chemical Society. (Dr. Georgiadis is affiliated with Indiana University− Purdue University Indianapolis, and Dr. Benner is affiliated with the Foundation for Applied Molecular Evolution, and Firebird Biomolecular Sciences.)

The first paper, “Structural Basis for a Six Nucleotide Genetic Alphabet”—appeared on May 11. It presented crystal structures to demonstrate that the new nucleotides paired with “geometries that are similar to those displayed by standard duplex DNA.” The second paper—“Evolution of Functional Six-Nucleotide DNA”—appeared a day later. It presented results form a laboratory in vitro evolution (LIVE) experiment to show that the new nucleotide bases could be incorporated into newly evolved, selectively binding species.

The first paper emphasized three important findings:

1. The discovery that canonical Watson–Crick pairing survives in duplexes that contain multiple and multiple adjacent Z:P pairs.
2. ZP-containing oligonucleotides adopt canonical helical forms, B- and A-form DNA. The ability of DNA to adopt A-form enables a number of important protein–DNA interactions such as those in the polymerase active site, which requires that the nucleobase pair within the active site and the adjacent pair adopt A-form in order to appropriately position the template-primer for optimal interactions with the polymerase increasing fidelity.
3. The Z-nitro group imparts new properties to the major groove of DNA that can potentially be exploited for recognition by proteins.

The second paper explained how the LIVE experiment involved developing a GACTZP library, which was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells.

“Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library,” the article’s authors wrote. “Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.”

Could CRISPR Be the Magic Bullet?

You Can Be “On Target” and Still Fail to Win a Prize

MaryAnn Labant

The research community’s rapid acceptance of the CRISPR/Cas technology is propelling a stage of deep investment in technology development. Already, three companies have emerged focusing on CRISPR therapeutic applications: Intellia Therapeutics, Editas Medicine, and CRISPR Therapeutics.

To continue to move the technology forward, scientists recently converged at the CRISPR Precision Gene Editing Congress to discuss unmet needs and new findings. The event, which took place in Boston, devoted particular attention to overcoming specificity, efficiency, and delivery challenges associated with the CRISPR/Cas9 system.

Many of these challenges relate to the mechanisms a cell may use to repair CRISPR/Cas-induced double-strand breaks (DSBs). A cell has two pathway choices. Non-homologous end joining (NHEJ), an error-prone ligation process, can result in small insertions and deletions (indels) at cleavage sites, whereas homology-directed repair (HDR) employs homologous DNA sequences as templates to make specific changes for precise repair. In most cells, NHEJ performs the majority of repair events.

Identifying and minimizing off-target events are major challenges. To meet these challenges, the Alt laboratory at Boston Children’s Hospital developed high-throughput genome translocation sequencing (HTGTS), an enzyme- and target-agnostic technique to rapidly expose potential off-target problems. Frederick W. Alt, Ph.D., and colleagues recently described the technique in an article that appeared in Nature Biotechnology.

“The method robustly detects DNA DSBs generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs,” the article read. “HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than 10-fold.”

When HTGTS was used to compare Cas9 nuclease and Cas9 paired nickases, paired nickases showed reduced off-target activity. Paired nickases were also assessed by scientists at Sigma-Aldrich.

“We compared paired nickases to Cas9-FokI nucleases. Paired nickases have about a 10-fold increase in design density, the number of nucleases that target a specific sequence in the selected area,” commented Gregory Davis, R&D manager, molecular biotechnology. “The more nuclease options, the better the chances of finding an active one near site-restricted locations such as disease single-nucleotide polymorphisms (SNPs).”

Like other companies, Sigma-Aldrich is evaluating methods to boost homologous recombination (HR) rates and inhibit NHEJ. Small molecules are being investigated, along with components of the DNA repair machinery such as mRNAs for RAD proteins. Enhancement techniques offer some improvement, but those improvements are not universally applicable to all cell types.

The company recently introduced a nuclease-based kinase knockout lentiviral library, but the challenge is increasing library screening effectiveness for cancer cell lines, which typically demonstrate some level of polyploidy. When the Cas9 nuclease library on the A459 lung cancer cell line was evaluated, a target diploid gene responded with a robust knockout, yet an expected knockout response for another gene was not seen. That particular gene turned out to be tetraploid.

Epigenetically based activators and inhibitors may be another approach, and the company is considering CRISPR-based gene regulation for inhibition or activation, CRISPRi or CRISPRa. Gene regulation may better simulate drugs that suppress activity and prove more effective than the nuclease-knockout method in lentiviral screening applications.

Measurement Systems

HDR and NHEJ editing events generally occur at low frequencies, necessitating ultrasensitive techniques for detection and quantification of edited alleles. While some studies have relied on NGS, a next-generation PCR technology called droplet digital PCR (ddPCR) is providing researchers with rapid, low-cost, ultrasensitive quantification of both NHEJ and HDR editing events.

ddPCR has already been widely used for high-sensitivity and high-precision applications such as rare cancer mutation detection and copy number analysis, noted Jennifer Berman, Ph.D., staff scientist, Digital Biology Center, Bio-Rad Laboratories.

Since HDR and NHEJ editing events can occur at very low frequency (<1%), especially HDR in primary or induced pluripotent stem (iPS) cells, ddPCR appears to be a fit for researchers wanting a rapid, sensitive, quantitative readout of editing in cells and tissues. The technique also enables empirical validation of guide RNA efficiency and measurement of the ratio of HDR:NHEJ at a targeted locus.

“ddPCR is one of the first sophisticated measurement systems for genome editing. The other option is sequencing, which is time-consuming, expensive and out of reach for most people,” explained Bruce Conklin, M.D., a senior investigator at the Gladstone Institute of Cardiovascular Disease and a professor of medicine at the University of California, San Francisco.

The Conklin laboratory works with iPS cells and is primarily interested in HDR, which is typically less than 1% of total alleles. A recent Nature Methods article by the group was the first demonstration that the genome could be changed one base at a time without any mark of an antibiotic reselection marker, a scarless replacement. Populations of cells that have a very rare cell with a single-base change are isolated using ddPCR as a measurement tool, then enriched sequentially, until a pure clone results, in a method termed sib-selection.

“With our method, you can see if the mutation you want is there from the start,” asserted Dr. Conklin. “Single base changes cause many human genetic diseases. To figure out the problem, you want to be able to change one thing and see what happens.”

“We are also looking at ddPCR to quantify HDR and NHEJ simultaneously to isolate conditions where there is more HDR than NHEJ,” he added. “Conditions are different in every cell type, for each location, and we do not understand the rules.”

Application to Animal Model

Mouse models have the potential to quickly screen and build a causal relationship between sequence variations in humans and their phenotypes. Historically, either pronuclear injection of a transgene into a mouse embryo or conventional gene targeting using embryonic stem (ES) cells produced new models.

In 2013, a study led by Rudolph Jaenisch, M.D., a professor of biology at MIT and a founding member of the Whitehead Institute for Biomedical Research, culminated in a published work that was the first to describe a CRISPR/Cas-engineered animal species. CRISPR’s ability to engineer targeted mutagenesis in the genome directly on the zygotes circumvents the need for germline-competent ES cells, and appears to result in more predictable models in a fraction of the previous time and cost.

The simplest way to create a knockout model is to inject CRISPR/Cas reagents, including Cas9 mRNA and a single gRNA, into the mouse embryo. If a knockin alteration is small, the intended mutation can be accommodated into a donor oligonucleotide of the maximal size of 200 bps; for larger alterations that cannot be engineered into a donor oligonucleotide, such as incorporation of a reporter gene or a human sequence, a donor plasmid is often used.

“We are now exploring the use of CRISPR for larger-scale genetic manipulation and humanization of the mouse genome. We do not know yet the size limitation of the genetic manipulation that you can introduce with the CRISPR/Cas technology,” discussed Wenning Qin, Ph.D., associate director of genetic engineering technologies, The Jackson Laboratory.

The Jackson Laboratory uses insertion of a fluorescent reporter gene into the Nanog locus, a gene expressed in early embryos, as the platform for parameter optimization. To determine if there were any off-target effects in addition to the on-target insertion of the reporter gene into the Nanog locus, two HDR mice carrying the reporter gene were genome sequenced, and evaluated minimally for the top 5,000 sites. No off-target effects were observed indicating that the CRISPR/Cas reagent used had a clean off-target profile among the examined sites.

Various means of enhancing the on-target efficiency of CRISPR/Cas9 modifications are being investigated by Taconic Biosciences. For example, the company is investigating the implementation of Cas9-orthologs to gain more flexibility in the choice of target sequences. It is also evaluating alternative delivery methods as well as experimental design and execution optimization.

To date, analyses have focused on evaluating on-target modifications. Nonetheless, various approaches to enhance specificity—for example, the use of Cas9 nickase, truncated sgRNAs, and Cas9 protein instead of mRNA—are being assessed. According to Jochen Welcker, Ph.D., senior manager of scientific development, improving the generation of more challenging types of alleles is a major development objective, and efficiency issues for specific applications need to be resolved.

For the generation of conditional knockout alleles, one obstacle is the efficient and correct integration of loxP-sites. Using ssODNs (single-stranded oligodeoxynucleotides) as donor material, for example, frequently leads to incomplete integration of the loxP-sites encoded by these ssODNs. The high efficiency of NHEJ-mediated deletion of the genomic region between the two loxP-sites is also a major hurdle as it leads to knockout alleles in up to 30% of the injected zygotes. The generation of complex knockin alleles is limited by the frequency of insertion/replacement of longer sequences by HDR.

Taconic has generated more than 200 humanized models by HDR. A drawback of this approach is the fact that only a single allelic variant can be modeled, while ever increasing amounts of human gene variants are being identified by genome-wide association studies (GWAS). Such variants can now be introduced quickly and efficiently, directly on existing humanized backgrounds by the use of CRISPR/Cas9 genome editing, making it possible to generate a whole range of human allelic variants from a single humanized-mouse model.

Bioinformatics Needs

“Bioinformatics plays an important role in CRISPR advancement. No-charge informatics packages are available, such as eCRISP, MIT tool, Doench activity scoring, and Zifit, that tackle a portion of the informatics aspects to CRISPR design, and many commercial companies have simple design tools built into their reagent-ordering systems,” stated Eric Rhodes, chief technology officer, Horizon Discovery. “Our new tool, gUIDEbook, a collaboration between Horizon and DesktopGenetics, is the first free application to combine all three aspects of informed design.”

First, all available protospacer adjacent motif (PAM) sites in a given region, which represent a potential gRNA design, must be found. Virtually all CRISPR design tools enable this and differ primarily in how the sequence is entered and user interface complexity. All programs essentially return the same information.

Second, off-target cutting potential by any given gRNA must be determined. Historically, searches found closely related sequences and scores were generated using weighting based on the number of mismatches, their location in the gRNA, and the number of occurrences in the genome. A new finding has demonstrated that “bulges” can occur in the matching of a gRNA to a potential target. More intensive searching is now required to identify all putative binding sites. The unknown is how likely an off-target identified by either method is actually going to be engaged.

The Doench algorithm, which is still early in its development, focuses solely on how much cutting activity a given gRNA design is likely to have. The algorithm calculates predictive scores on the basis of known guides and cutting activities. It does not account for off-target potential, so it has somewhat limited standalone value.

Delivery

Thermo Fisher Scientific supplies a complete workflow for gene editing and cell engineering that focuses on design, delivery, and analysis. Transfection-grade Cas9 protein and mRNA have been functionally tested in several cell lines, including iPS and ES cells, and both contain a nuclear localization signal (NLS) to aid in delivery. The GeneArt Cas9 Nuclease is extensively purified and quality controlled to remove nonspecific endonucleases and endotoxins.

According to Jason Potter, senior scientist of protein engineering, cell lines vary in how easily they can be transfected. With plasmids and mRNA, the cell must still process the transcripts and make Cas9 complexes before it can act. To simplify the process, the gRNA can be made and complexed with the transfection-grade Cas9 protein in vitro. After it is delivered by lipids or electroporation is used, the Cas9 complex is able to act once it reaches the nucleus. Analysis of the edited cells can then be done using the GeneArt Genomic Cleavage Detection kit or by sequencing.

Lipids, including Lipofectamine 3000, Lipofectamine RNAiMAX, and Lipofectamine MessengerMAX, have been used for delivery of plasmids and RNAs for years. Drawing on this knowledge of Cas9, the company has optimized lipid dosages and protocols for high transfection efficiency and low toxicity. Due to the exposed guide RNA component of the Cas9 complex, RNAiMAX also works for delivery of Cas9 protein. For electroporation, the key consideration is optimizing the voltage and pulse conditions for the cell line.

Gene Editing with Cas9 and Optimal Promoter

The Cas9 (CRISPR associated protein 9) system has gained significant interest due to its relative simplicity and ease of use compared to other genome-engineering technologies, according to many scientists. The CRISPR/Cas9 system requires a complex of the Cas9 protein with a trans-activating RNA (tracrRNA) and a gene-targeting CRISPR RNA (crRNA) or a single guide RNA (sgRNA, a chimeric form of tracrRNA with a crRNA).

Researchers at Dharmacon, now part of GE Healthcare, recently carried out a study on the efficiency of using synthetic crRNA and tracrRNA to introduce gene-editing events when co-transfected with a plasmid expressing Cas9. They explored the use of antibiotic and FACS methods for enrichment of cells that have undergone gene editing, and the use of multiple promoters to increase efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNA.

The researchers reported that utilizing a highly active promoter for Cas9 expression enables better editing in specific cell lines. Enrichment of transiently transfected cells either by fluorescence-activated cell soring or puromycin selection can further improve the yield of edited cells, they added.

In addition, they concluded that efficient gene editing can be achieved with a three-component system: plasmid Cas9 and synthetic tracrRNA and crRNA. They also pointed out that use of synthetic tracrRNA and crRNA is a simplified method for gene editing of one or more genes without requiring any cloning steps, and that the three-component CRISPR/Cas9 system is amenable to high-throughput genome editing applications.

New Stem Cell Identified

By Anna Azvolinsky | May 6, 2015

Researchers isolate an easy-to-manipulate, stable, and spatially distinct pluripotent cell type.

Scientists have isolated and defined a new type of pluripotent cell from early mouse embryos and from monkey and human stem cell lines. The monkey- and human-derived versions of these pluripotent cells can divide and generate the three germ layers in a developing mouse embryo, providing the first demonstration that human pluripotent cells can begin a differentiation program inside mice. In their May 6 Nature paper reporting these results, developmental biologist Juan Carlos Izpisua Belmonte of the Salk Institute for Biological Studies in La Jolla, California, and his colleagues suggested that these newly identified cells may be useful for modeling early human development and might in the future be used to generate tissues and organs for clinical applications.

“I found the paper fascinating,” said George Daley, a stem cell biologist at Children’s Hospital Boston and Harvard Medical School who was not involved in the work. “It highlights a new type of pluripotent stem cell, which is among the most exciting aspects of stem cell biology over the last several years.”

The findings, said Paul Knoepfler, a stem cell biologist at the University of California, Davis, who was not involved in the work, “fit nicely into a broader concept that there are going to be ‘intermediate state’ stem cells that don’t fit so easily into binary, black-and-white ways of classifying [pluripotent cells].”

These so-called novel region-selective pluripotent stem cells (rsPSCs) are further differentiated compared to previously isolated stem cells and have more favorable characteristics for laboratory manipulation, including high cloning efficiency, stable passage in culture, and ease of genetic engineering. “All of these are advantages for applications in research and, potentially, scaling up for clinical applications,” said Daley.

RsPSCs are distinct from two other pluripotent mouse cell types that researchers have been able to stably propagate in culture—naive embryonic stem cells (ESCs), from the pre-implantation embryo, and primed epiblast stem cells (EpiSCs), derived from the post-implantation embryo.

“We believe that [rsPSCs] should have the ability to differentiate more efficiently into somatic cells . . . because they are right at the junction of pluripotency and differentiation,” said study coauthor Jun Wu, a research associate in the Belmonte lab.

In the post-implantation mouse embryo, cells are already distinguished into anterior, posterior, distal, and proximal functional orientations. The researchers sought to determine whether they could capture a cell type from the post-implantation embryo with distinct properties conferred by one of these spatial orientations. The team focused on isolating and culturing posterior cells where gastrulation occurs in the early embryo.

Wu and his colleagues found that a combination of serum-free media plus fibroblast growth factor 2 (FGF2) and Wnt signaling inhibitors resulted in stable rsPSCs.

The transcriptomes of these cells resembled those of the posterior cells of the early mouse embryo, and grafting these cells into 7.5-day-old mouse embryos resulted in efficient incorporation in the posterior, but not the other parts of the embryo. After 36 hours of culturing these chimaeric embryos, the rsPSCs proliferated and could differentiate into the developing three germ layers. The results of this 36-hour embryo incubation assay suggest that rsPSCs have the potential to fulfill their developmental program in vivo, said Jun.

A distinguishing feature of naive ESCs compared with primed EpiSCs is their ability to efficiently form colonies from single cells—a trait called high clonagenicity. Yet even though rsPSCs resemble cells from post-implantation embryos, just like the EpiSCs, these cells readily formed clones. RsPSCs also produced smaller teratomas—tumors that resemble embryos, including tissues from all three germ layers—when injected into mice. And while conventional human ESCs could not, human rsPSCs could also be grafted into the mouse embryo and form all three germ layers.

“The high clonal efficiency was surprising, as was the data that these cells form smaller teratoma,” said Knoepfler. “Anything that forms a smaller teratoma would have a potentially better safety profile for clinical applications,” he added.

The authors notes that human and nonhuman primate ESC lines could be used to create rsPSCs with similar characteristics to the mouse cells, including improved cloning efficiency.

Because of the ethical limitations of working with human embryos, the standard functional test for pluripotency of human stem cells is the mouse teratoma assay. “We are excited that this is the first demonstration that [human stem cells] can enter into the early developmental program of another species,” said Jun.

Whether human rsPSCs can generate more complicated tissue structures within mice or other animals requires further study, Belmonte told The Scientist via e-mail. “Of course,” he noted, “the ethical implications behind creating a human-animal chimera for the purpose of obtaining human tissues and organs to save lives of millions needs to be carefully evaluated.” 

Daley pointed out that this study is not the first to describe a novel pluripotency state. But these studies have been the subject of some controversy because the cells have been unstable in culture, and independent laboratories have struggled to reproduce the reported results.

“It remains to be seen whether these conditions for rsPSCs are reproducible, [but] it looks like [the authors] were very effective in isolating these cells,” he said. “The fact that this works across the spectrum of mammals suggests that it is a pretty stable state.”That changing culture conditions can generate different kinds of stem cells is not surprising, said Knoepfler, but it is exciting. “I expect that more new cell types will be found in the future,” he said.

J. Wu et al., “An alternative pluripotent state confers interspecies chimaeric competency,”Nature, doi:10.1038/nature14413, 2015.