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/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4

Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine

Complete Genome Sequences of T4-Like Bacteriophages RB3, RB5, RB6, RB7, RB9, RB10, RB27, RB33, RB55, RB59, and RB68

T4-like bacteriophages have been explored for phage therapy and are model organisms for phage genomics and evolution. Here, we describe the sequencing of 11 T4-like phages. We found a high nucleotide similarity among the T4, RB55, and RB59; RB32 and RB33; and RB3, RB5, RB6, RB7, RB9, and RB10 phages

Concerning RNA-guided gene drives for the alteration of wild populations

Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology. DOI: http://dx.doi.org/10.7554/eLife.03401.00

Can large language models democratize access to dual-use biotechnology?

Large language models (LLMs) such as those embedded in 'chatbots' are accelerating and democratizing research by providing comprehensible information and expertise from many different fields. However, these models may also confer easy access to dual-use technologies capable of inflicting great harm. To evaluate this risk, the 'Safeguarding the Future' course at MIT tasked non-scientist students with investigating whether LLM chatbots could be prompted to assist non-experts in causing a pandemic. In one hour, the chatbots suggested four potential pandemic pathogens, explained how they can be generated from synthetic DNA using reverse genetics, supplied the names of DNA synthesis companies unlikely to screen orders, identified detailed protocols and how to troubleshoot them, and recommended that anyone lacking the skills to perform reverse genetics engage a core facility or contract research organization. Collectively, these results suggest that LLMs will make pandemic-class agents widely accessible as soon as they are credibly identified, even to people with little or no laboratory training. Promising nonproliferation measures include pre-release evaluations of LLMs by third parties, curating training datasets to remove harmful concepts, and verifiably screening all DNA generated by synthesis providers or used by contract research organizations and robotic cloud laboratories to engineer organisms or viruses.Comment: 6 pages, 0 figure

Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing

The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but 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 identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells

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

CRISPR-Cas systems have been used with single-guide RNAs for accurate gene disruption and conversion in multiple biological systems. Here we report the use of the endonuclease Cas9 to target genomic sequences in the C. elegans germline, utilizing 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

BIOSAFETY. Safeguarding gene drive experiments in the laboratory.

Multiple stringent confinement strategies should be used whenever possibleThis is the author accepted manuscript. The final version is available from AAAS via http://dx.doi.org/10.1126/science.aac793

Genetic frontiers for conservation:An assessment of synthetic biology and biodiversity conservation

In recent years synthetic biology has emerged as a suite of techniques and technologies that enable humans to read, interpret, modify, design and manufacture DNA in order to rapidly influence the forms and functions of cells and organisms, with the potential to reach whole species and ecosystems. As synthetic biology continues to evolve, new tools emerge, novel applications are proposed, and basic research is applied. This assessment is one part of IUCN’s effort to provide recommendations and guidance regarding the potential positive and negative impacts of synthetic biology on biodiversity conservation; it comprises a full assessment and a short synthesis report

A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system