Systematic molecular evolution enables robust biomolecule discovery.

Evolution occurs when selective pressures from the environment shape inherited variation over time. Within the laboratory, evolution is commonly used to engineer proteins and RNA, but experimental constraints have limited the ability to reproducibly and reliably explore factors such as population diversity, the timing of environmental changes and chance on outcomes. We developed a robotic system termed phage- and robotics-assisted near-continuous evolution (PRANCE) to comprehensively explore biomolecular evolution by performing phage-assisted continuous evolution in high-throughput. PRANCE implements an automated feedback control system that adjusts the stringency of selection in response to real-time measurements of each molecular activity. In evolving three distinct types of biomolecule, we find that evolution is reproducibly altered by both random chance and the historical pattern of environmental changes. This work improves the reliability of protein engineering and enables the systematic analysis of the historical, environmental and random factors governing biomolecular evolution.

Low-N protein engineering with data-efficient deep learning.

Protein engineering has enormous academic and industrial potential. However, it is limited by the lack of experimental assays that are consistent with the design goal and sufficiently high throughput to find rare, enhanced variants. Here we introduce a machine learning-guided paradigm that can use as few as 24 functionally assayed mutant sequences to build an accurate virtual fitness landscape and screen ten million sequences via in silico directed evolution. As demonstrated in two dissimilar proteins, GFP from Aequorea victoria (avGFP) and E. coli strain TEM-1 ß-lactamase, top candidates from a single round are diverse and as active as engineered mutants obtained from previous high-throughput efforts. By distilling information from natural protein sequence landscapes, our model learns a latent representation of 'unnaturalness', which helps to guide search away from nonfunctional sequence neighborhoods. Subsequent low-N supervision then identifies improvements to the activity of interest. In sum, our approach enables efficient use of resource-intensive high-fidelity assays without sacrificing throughput, and helps to accelerate engineered proteins into the fermenter, field and clinic.

Insidious Insights: Implications of viral vector engineering for pathogen enhancement.

Optimizing viral vectors and their properties will be important for improving the effectiveness and safety of clinical gene therapy. However, such research may generate dual-use insights relevant to the enhancement of pandemic pathogens. In particular, reliable and generalizable methods of immune evasion could increase viral fitness sufficient to cause a new pandemic. High potential for misuse is associated with (1) the development of universal genetic elements for immune modulation, (2) specific insights on capsid engineering for antibody evasion applicable to viruses with pandemic potential, and (3) the development of computational methods to inform capsid engineering. These risks may be mitigated by prioritizing non-viral delivery systems, pharmacological immune modulation methods, non-genetic vector surface modifications, and engineering methods specific to AAV and other viruses incapable of unassisted human-to-human transmission. We recommend that computational vector engineering and the publication of associated code and data be limited to AAV until a technical solution for preventing malicious access to viral engineering tools has been established.

Enabling high-throughput biology with flexible open-source automation.

Our understanding of complex living systems is limited by our capacity to perform experiments in high throughput. While robotic systems have automated many traditional hand-pipetting protocols, software limitations have precluded more advanced maneuvers required to manipulate, maintain, and monitor hundreds of experiments in parallel. Here, we present Pyhamilton, an open-source Python platform that can execute complex pipetting patterns required for custom high-throughput experiments such as the simulation of metapopulation dynamics. With an integrated plate reader, we maintain nearly 500 remotely monitored bacterial cultures in log-phase growth for days without user intervention by taking regular density measurements to adjust the robotic method in real-time. Using these capabilities, we systematically optimize bioreactor protein production by monitoring the fluorescent protein expression and growth rates of a hundred different continuous culture conditions in triplicate to comprehensively sample the carbon, nitrogen, and phosphorus fitness landscape. Our results demonstrate that flexible software can empower existing hardware to enable new types and scales of experiments, empowering areas from biomanufacturing to fundamental biology.

Daisy-chain gene drives for the alteration of local populations.

If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. "Daisy-drive" systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.

Inoculating science against potential pandemics and information hazards.

The recent de novo assembly of horsepox is an instructive example of an information hazard: published methods enabling poxvirus synthesis led to media coverage spelling out the implications, efficiently disseminating true information that might be used to cause harm. Whether or not the benefits justified the risks, the horsepox saga provides ample reason to upgrade the current system for screening synthesized DNA for hazardous sequences, which does not cover the majority of firms and cannot reliably prevent the assembly of potentially pandemic pathogens. An upgraded system might leverage one-way encryption to confidentially scrutinize virtually all commercial production by a cooperative international network of servers whose integrity can be verified by third parties. Funders could support participating institutions to ease the transition or outright subsidize the market to make clean DNA cheaper, while boycotts by journals, institutions, and funders could ensure compliance and require hardware-level locks on future DNA synthesizers. However, the underlying problem is that security and safety discussions among experts typically follow potentially hazardous events rather than anticipating them. Changing norms and incentives to favor preregistration and advisory peer review of planned experiments could test alternatives to the current closeted research model in select areas of science. Because the fields of synthetic mammalian virology and especially gene drive research involve technologies that could be unilaterally deployed and may self-replicate in the wild, they are compelling candidates for initial trials of early-stage peer review.

Conservation demands safe gene drive.

Interest in developing gene drive systems to control invasive species is growing, with New Zealand reportedly considering the nascent technology as a way to locally eliminate the mammalian pests that threaten its unique flora and fauna. If gene drives successfully eradicated these invasive populations, many would rejoice, but what are the possible consequences? Here, we explore the risk of accidental spread posed by self-propagating gene drive technologies, highlight new gene drive designs that might achieve better outcomes, and explain why we need open and international discussions concerning a technology that could have global ramifications.

A system for the continuous directed evolution of biomolecules.

Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention. Because evolutionary success is dependent on the total number of rounds performed, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. Although researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Wright and Joyce, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in Escherichia coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerase (RNAP) variants that recognize a distinct promoter, initiate transcripts with ATP instead of GTP, and initiate transcripts with CTP. In one example, PACE executed 200 rounds of protein evolution over the course of 8 days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than 1 week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

Cas9 as a versatile tool for engineering biology.

RNA-guided Cas9 nucleases derived from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have dramatically transformed our ability to edit the genomes of diverse organisms. We believe tools and techniques based on Cas9, a single unifying factor capable of colocalizing RNA, DNA and protein, will grant unprecedented control over cellular organization, regulation and behavior. Here we describe the Cas9 targeting methodology, detail current and prospective engineering advances and suggest potential applications ranging from basic science to the clinic.

Heritable genome editing in C. elegans via a CRISPR-Cas9 system.

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.

The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.

Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution.

To what extent are evolutionary outcomes determined by a population's recent environment, and to what extent do they depend on historical contingency and random chance? Here we apply a unique experimental system to investigate evolutionary reproducibility and path dependence at the protein level. We combined phage-assisted continuous evolution with high-throughput sequencing to analyze evolving protein populations as they adapted to divergent and then convergent selection pressures over hundreds of generations. Independent populations of T7 RNA polymerase genes were subjected to one of two selection histories ("pathways") demanding recognition of distinct intermediate promoters followed by a common final promoter. We observed distinct classes of solutions with unequal phenotypic activity and evolutionary potential evolve from the two pathways, as well as from replicate populations exposed to identical selection conditions. Mutational analysis revealed specific epistatic interactions that explained the observed path dependence and irreproducibility. Our results reveal in molecular detail how protein adaptation to different environments, as well as stochasticity among populations evolved in the same environment, can both generate evolutionary outcomes that preclude subsequent convergence.

Genome-scale engineering for systems and synthetic biology.

Genome-modification technologies enable the rational engineering and perturbation of biological systems. Historically, these methods have been limited to gene insertions or mutations at random or at a few pre-defined locations across the genome. The handful of methods capable of targeted gene editing suffered from low efficiencies, significant labor costs, or both. Recent advances have dramatically expanded our ability to engineer cells in a directed and combinatorial manner. Here, we review current technologies and methodologies for genome-scale engineering, discuss the prospects for extending efficient genome modification to new hosts, and explore the implications of continued advances toward the development of flexibly programmable chasses, novel biochemistries, and safer organismal and ecological engineering.

CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males.

Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

PyLabRobot: An Open-Source, Hardware Agnostic Interface for Liquid-Handling Robots and Accessories.

Liquid handling robots are often limited by proprietary programming interfaces that are only compatible with a single type of robot and operating system, restricting method sharing and slowing development. Here we present PyLabRobot, an open-source, cross-platform Python interface capable of programming diverse liquid-handling robots, including Hamilton STARs, Tecan EVOs, and Opentron OT-2s. PyLabRobot provides a universal set of commands and representations for deck layout and labware, enabling the control of diverse accessory devices. The interface is extensible and can work with any robot that manipulates liquids within a Cartesian coordinate system. We validated the system through unit tests and several application demonstrations, including a browser-based simulator, a position calibration tool, and a path-teaching tool for complex movements. PyLabRobot provides a flexible, open, and collaborative programming environment for laboratory automation.

CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males.

Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile Insect Technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.

Measuring the tolerance of the genetic code to altered codon size.

Translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet codon, rather than quadruplet codon, genetic code? Here, we investigate the tolerance of the Escherichia coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all 20 canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs). Many of these selectively incorporate a single amino acid in response to a specified four-base codon, as confirmed with mass spectrometry. However, efficient quadruplet codon translation often requires multiple tRNA mutations. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These may constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results suggest that the triplet codon code was selected because it is simpler and sufficient, not because a quadruplet codon code is unachievable. These data provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

Analysis of the first genetic engineering attribution challenge.

The ability to identify the designer of engineered biological sequences-termed genetic engineering attribution (GEA)-would help ensure due credit for biotechnological innovation, while holding designers accountable to the communities they affect. Here, we present the results of the first Genetic Engineering Attribution Challenge, a public data-science competition to advance GEA techniques. Top-scoring teams dramatically outperformed previous models at identifying the true lab-of-origin of engineered plasmid sequences, including an increase in top-1 and top-10 accuracy of 10 percentage points. A simple ensemble of prizewinning models further increased performance. New metrics, designed to assess a model's ability to confidently exclude candidate labs, also showed major improvements, especially for the ensemble. Most winning teams adopted CNN-based machine-learning approaches; however, one team achieved very high accuracy with an extremely fast neural-network-free approach. Future work, including future competitions, should further explore a wide diversity of approaches for bringing GEA technology into practical use.

Direct and indirect impacts of synthetic biology on biodiversity conservation.

The world's biodiversity is in crisis. Synthetic biology has the potential to transform biodiversity conservation, both directly and indirectly, in ways that are negative and positive. However, applying these biotechnology tools to environmental questions is fraught with uncertainty and could harm cultures, rights, livelihoods, and nature. Decisions about whether or not to use synthetic biology for conservation should be understood alongside the reality of ongoing biodiversity loss. In 2022, the 196 Parties to the United Nations Convention on Biological Diversity are negotiating the post-2020 Global Biodiversity Framework that will guide action by governments and other stakeholders for the next decade to conserve the worlds' biodiversity. To date, synthetic biologists, conservationists, and policy makers have operated in isolation. At this critical time, this review brings these diverse perspectives together and emerges out of the need for a balanced and inclusive examination of the potential application of these technologies to biodiversity conservation.