SynBioFromLeukipposInstitute

Nature is a diverse and rich source of bioactive pathways or novel building blocks for synthetic biology. In this Perspective, we describe the emerging research field in which metagenomes are functionally interrogated using synthetic biology. This approach substantially expands the set of identified biological activities and building blocks. In reviewing this field, we find that its potential for new biological discovery is dramatically increasing. Functional metagenomic mining using genetic circuits has led to the discovery of novel bioactivity such as amidases, NF-κB modulators, naphthalene degrading enzymes, cellulases, lipases and transporters. Using these genetic circuits as a template, improvements are made by designing biosensors, such as in vitro–evolved riboswitches and computationally redesigned transcription factors. Thus, powered by the rapidly expanding repertoire of biosensors and streamlined processes for automated genetic circuit design, a greater variety of complex selection circuits can be built, with resulting impacts on drug discovery and industrial biotechnology.

A common theme in the self-organization of multicellular tissues is the use of cell-cell signaling networks to induce morphological changes. We used the modular synNotch juxtacrine signaling platform to engineer artificial genetic programs in which specific cell-cell contacts induced changes in cadherin cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems: robust self-organization into multidomain structures, well-choreographed sequential assembly, cell type divergence, symmetry breaking, and the capacity for regeneration upon injury. The ability of these networks to drive complex structure formation illustrates the power of interlinking cell signaling with cell sorting: Signal-induced spatial reorganization alters the local signals received by each cell, resulting in iterative cycles of cell fate branching. These results provide insights into the evolution of multicellularity and demonstrate the potential to engineer customized self-organizing tissues or materials.

From bacteria following simple chemical gradients1 to the brain distinguishing complex odour information2, the ability to recognize molecular patterns is essential for biological organisms. This type of information-processing function has been implemented using DNA-based neural networks3, but has been limited to the recognition of a set of no more than four patterns, each composed of four distinct DNA molecules. Winner-take-all computation4 has been suggested5,6 as a potential strategy for enhancing the capability of DNA-based neural networks. Compared to the linear-threshold circuits7 and Hopfield networks8 used previously3, winner-take-all circuits are computationally more powerful4, allow simpler molecular implementation and are not constrained by the number of patterns and their complexity, so both a large number of simple patterns and a small number of complex patterns can be recognized. Here we report a systematic implementation of winner-take-all neural networks based on DNA-strand-displacement9,10 reactions. We use a previously developed seesaw DNA gate motif3,11,12, extended to include a simple and robust component that facilitates the cooperative hybridization13 that is involved in the process of selecting a ‘winner’. We show that with this extended seesaw motif DNA-based neural networks can classify patterns into up to nine categories. Each of these patterns consists of 20 distinct DNA molecules chosen from the set of 100 that represents the 100 bits in 10 × 10 patterns, with the 20 DNA molecules selected tracing one of the handwritten digits ‘1’ to ‘9’. The network successfully classified test patterns with up to 30 of the 100 bits flipped relative to the digit patterns ‘remembered’ during training, suggesting that molecular circuits can robustly accomplish the sophisticated task of classifying highly complex and noisy information on the basis of similarity to a memory.

Synthetic biology applies engineering concepts to build cells that perceive and process information. Examples include cells engineered to perform basic digital or analog computation. These circuits serve as basis for the construction of complex integrated cellular networks that offer manifold applications in fundamental and applied research. Here, we introduce the concept of using design approaches and molecular tools applied in synthetic biology for the construction of interconnected biohybrid materials systems with information processing functionality. We validate this concept by modularly assembling protein and polymer building blocks to generate stimulus-responsive materials. Guided by a quantitative mathematical model, we next interconnect these materials into a materials system that acts as both a signal detector and as an amplifier based on a built-in positive feedback loop. The functionality and versatility of this materials system is demonstrated by the detection of enzymatic activities and drugs. The modular design concept presented here thus represents a blueprint for integrating synthetic biology-inspired information-processing circuits into polymer materials. As integrated sensors and actuators, the resulting smart materials systems could provide novel solutions with broad perspectives in research and development.

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP–ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein–DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1,2,3,4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.

Researchers from the University of Illinois at Chicago are the first to describe why CRISPR gene editing sometimes fails to work, and how the process can be made to be much more efficient.
CRISPR is a gene-editing tool that allows scientists to cut out unwanted genes or genetic material from DNA, and sometimes add a desired sequence or genes. CRISPR uses an enzyme called Cas9 that acts like scissors to cut out unwanted DNA. Once cuts are made on either side of the DNA to be removed, the cell either initiates repair to glue the two ends of the DNA strand back together, or the cell dies.
In a study published in the journal Molecular Cell, the researchers showed that when gene editing using CRISPR fails, which occurs about 15 percent of the time, it is often due to persistent binding of the Cas9 protein to the DNA at the cut site, which blocks the DNA repair enzymes from accessing the cut.
Senior author Bradley Merrill, associate professor of biochemistry and molecular genetics in the UIC College of Medicine, says that before now, researchers did not know why the process randomly failed.
"We found that at sites where Cas9 was a 'dud' it stayed bound to the DNA strand and prevented the cell from initiating the repair process," Merrill said. The stuck Cas9 is also unable to go on to make additional cuts in DNA, thus limiting the efficiency of CRISPR, he said.
Merrill, UIC graduate student Ryan Clarke, and their colleagues also found that Cas9 was likely to be ineffective at sites in the genome where RNA polymerases—enzymes involved in gene activity—were not active. Further investigation revealed that guiding Cas9 to anneal to just one of the strands making up the DNA double helix promoted interaction between Cas9 and the RNA polymerase, helping to transform a "dud" Cas9 into an efficient genome editor.
Specifically, they found that consistent strand selection for Cas9 during genome editing forced the RNA polymerases to collide with Cas9 in such a way that Cas9 was knocked off the DNA.
"I was shocked that simply choosing one DNA strand over the other had such a powerful effect on genome editing," said Clarke, the lead author of the paper. "Uncovering the mechanism behind this phenomenon helps us better understand how Cas9 interactions with the genome can cause some editing attempts to fail and that, when designing a genome editing experiment, we can use that understanding to our benefit."
"This new understanding is important for those of us who need genome editing to work well in the lab and for making genome editing more efficient and safer in future clinical uses," Merrill said.
The study findings are also significant because, in the genome editing process, the interaction between Cas9 and the DNA strand is now known to be the "rate-limiting step," said Merrill. This means that it is the slowest part of the process; therefore, changes at this stage have the most potential to impact the overall duration of genome editing.
"If we can reduce the time that Cas9 interacts with the DNA strand, which we now know how to do with an RNA polymerase, we can use less of the enzyme and limit exposure," Merrill said. "This means we have more potential to limit adverse effects or side effects, which is vital for future therapies that may impact human patients."
Explore further: Genome-editing tool could increase cancer risk
More information: Ryan Clarke et al, Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks, Molecular Cell (2018). DOI: 10.1016/j.molcel.2018.06.005

Synthetic genetic circuits can induce cells to form simple 3D structures reminiscent of those generated during early embryonic development. This advance will help engineers build tissues that have desirable structures.