Scientists Hack a Human Cell and Reprogram It Like a Computer

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CELLS ARE BASICALLY tiny computers: They send and receive inputs and output accordingly. If you chug a Frappuccino, your blood sugar spikes, and your pancreatic cells get the message. Output: more insulin.

But cellular computing is more than just a convenient metaphor. In the last couple of decades, biologists have been working to hack the cells’ algorithm in an effort to control their processes. They’ve upended nature’s role as life’s software engineer, incrementally editing a cell’s algorithm—its DNA—over generations. In a paper published today in Nature Biotechnology, researchers programmed human cells to obey 109 different sets of logical instructions. With further development, this could lead to cells capable of responding to specific directions or environmental cues in order to fight disease or manufacture important chemicals.

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells

Benjamin H Weinberg, N T Hang Pham, Leidy D Caraballo, Thomas Lozanoski, Adrien Engel, Swapnil Bhatia & Wilson W Wong
Affiliations Contributions Corresponding author

Nature Biotechnology 35, 453–462 (2017) 

doi:10.1038/nbt.3805
Received 20 June 2016
Accepted 27 January 2017
Published online 27 March 2017

Engineered genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. We present a robust, general, scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE), to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on recombinases under the control of a single promoter, which integrates circuit signals on a single transcriptional layer. We used BLADE to build 113 circuits in human embryonic kidney and Jurkat T cells and devised a quantitative, vector-proximity metric to evaluate their performance. Of 113 circuits analyzed, 109 functioned (96.5%) as intended without optimization. The circuits, which are available through Addgene, include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; circuits with small-molecule-inducible control; and circuits that incorporate CRISPR–Cas9 to regulate endogenous genes. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering.

Their cells execute these instructions by using proteins called DNA recombinases, which cut, reshuffle, or fuse segments of DNA. These proteins recognize and target specific positions on a DNA strand—and the researchers figured out how to trigger their activity. Depending on whether the recombinase gets triggered, the cell may or may not produce the protein encoded in the DNA segment.

A cell could be programmed, for example, with a so-called NOT logic gate. This is one of the simplest logic instructions: Do NOT do something whenever you receive the trigger. This study’s authors used this function to create cells that light up on command. Biologist Wilson Wong of Boston University, who led the research, refers to these engineered cells as “genetic circuits.”

Here’s how it worked: Whenever the cell did contain a specific DNA recombinase protein, it would NOT produce a blue fluorescent protein that made it light up. But when the cell did not contain the enzyme, its instruction was DO light up. The cell could also follow much more complicated instructions, like lighting up under longer sets of conditions.

Wong says that you could use these lit up cells to diagnose diseases, by triggering them with proteins associated with a particular disease. If the cells light up after you mix them with a patient’s blood sample, that means the patient has the disease. This would be much cheaper than current methods that require expensive machinery to analyze the blood sample.

Now, don’t get distracted by the shiny lights quite yet. The real point here is that the cells understand and execute directions correctly. “It’s like prototyping electronics,” says biologist Kate Adamala of the University of Minnesota, who wasn’t involved in the research. As every Maker knows, the first step to building complex Arduino circuits is teaching an LED to blink on command.

Pharmaceutical companies are teaching immune cells to be better cancer scouts using similar technology. Cancer cells have biological fingerprints, such as a specific type of protein. Juno Therapeutics, a Seattle-based company, engineers immune cells that can detect these proteins and target cancer cells specifically. If you put logic gates in those immune cells, you could program the immune cells to destroy the cancer cells in a more sophisticated and controlled way.

Programmable cells have other potential applications. Many companies use genetically modified yeast cells to produce useful chemicals. Ginkgo Bioworks, a Boston-based company, uses these yeast cells to produce fragrances, which they have sold to perfume companies. This yeast eats sugar just like brewer’s yeast, but instead of producing alcohol, it burps aromatic molecules. The yeast isn’t perfect yet: Cells tend to mutate as they divide, and after many divisions, they stop working well. Narendra Maheshri, a scientist at Ginkgo, says that you could program the yeast to self-destruct when it stops functioning properly, before they spoil a batch of high-grade cologne.

Wong’s group wasn’t the first to make biological logic gates, but they’re the first to build so many with consistent success. Of the 113 circuits they built, 109 worked. “In my personal experience building genetic circuits, you’d be lucky if they worked 25 percent of the time,” Wong says. Now that they’ve gotten these basic genetic circuits to work, the next step is to make the logic gates work in different types of cells.

But it won’t be easy. Cells are incredibly complicated—and DNA doesn’t have straightforward “on” and “off” switches like an electronic circuit. In Wong’s engineered cells, you “turn off” the production of a certain protein by altering the segment of DNA that encodes its instructions. It doesn’t always work, because nature might have encoded some instructions in duplicate. In other words: It’s hard to debug 3 billion years of evolution.

ORIGINAL: Wired

By SOPHIA CHEN.

03.27.17

Could CRISPR Be the Magic Bullet?

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

Research scientists and tool suppliers in the life sciences continue to devote resources to CRISPR, which is still a relatively new tool. Much is still unknown, and the community needs a deeper understanding of the technology to better harness CRISPR for discovery and development as well as eventual clinical applications. [Thermo Fisher Scientific]

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
• 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.

1. Non-homologous end joining (NHEJ), an error-prone ligation process, can result in small insertions and deletions (indels) at cleavage sites, whereas 
2. 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.”

Sigma-Aldrich research scientists Greg Davis, Ph.D., and Fuqiang Chen, Ph.D., discuss genome-editing technologies and best practices.

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

Droplet digital PCR (ddPCR), a next-generation polymerase chain reaction technology from Bio-Rad Laboratories, can provide rapid, low-cost, ultra-sensitive quantification of both NHEJ- and HDR-editing events.

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 Models

The Jackson Laboratory advises that the simplest way to create a knockout mouse model is to inject CRISPR/Cas reagents, including Cas9 mRNA and a single gRNA, into a mouse embryo.

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.

ORIGINAL: Genetic Engineering & Biotechnology News

By MaryAnn Labant

May 1, 2015 (Vol. 35, No. 9)